SATINSKY BRANDON M (US)
BASU SHIB (US)
US6681186B1 | 2004-01-20 |
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CLAIMS 1. A synthetic composition, comprising one or more endophytes heterologously disposed to a treatment formulation, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO.127, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.128-131. 2. The synthetic composition of Claim 1, wherein the synthetic composition additionally comprises one or more plant element elements. 3. The synthetic composition of Claim 2, wherein the one or more plant element elements are seeds. 4. The synthetic composition of Claim 2, wherein the one or more plant element elements are soybean, wheat, or corn. 5. The synthetic composition of Claim 3, wherein the synthetic composition comprises a least 1E+02 endophytes per seed. 6. The synthetic composition of Claim 3, wherein the synthetic composition comprises a least 1E+03 endophytes per seed. 7. The synthetic composition of Claim 3, wherein the synthetic composition comprises a least 1E+04 endophytes per seed. 8. The synthetic composition of Claim 2, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 9. The synthetic composition of Claim 8, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, yield, plant height, shoot weight, and or root weight. 10. The synthetic composition of Claim 9, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 11. The synthetic composition of Claim 10, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 12. The synthetic composition of Claim 8, wherein: the synthetic composition additionally comprises one or more soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium, Rhizoctonia, or Fusarium species. 13. The synthetic composition of Claim 8, wherein: the synthetic composition additionally comprises one or more wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 14. The synthetic composition of Claim 8, wherein: the synthetic composition additionally comprises one or more corn plant elements, the one or more traits of agronomic importance are shoot weight, yield, plant height, root weight, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 15. The synthetic composition of Claim 1, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.127. 16. The synthetic composition of Claim 1, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs.128, 129, 130, and 131. 17. The synthetic composition of Claim 1, wherein the one or more endophytes are of the genus Chitinophaga. 18. The synthetic composition of Claim 1, wherein the one or more endophytes are of the genus and species Chitinophaga oryzae. 19. A synthetic composition, comprising one or more endophytes heterologously disposed to a treatment formulation, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO.132, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.133-136, wherein the treatment formulation comprises a solid carrier and adherent.. 20. The synthetic composition of Claim 19, wherein the synthetic composition additionally comprises one or more plant element elements. 21. The synthetic composition of Claim 20, wherein the one or more plant element elements are seeds. 22. The synthetic composition of Claim 20, wherein the one or more plant element elements are soybean, wheat, or cotton. 23. The synthetic composition of Claim 21, wherein the synthetic composition comprises a least 1E+02 endophytes per seed. 24. The synthetic composition of Claim 21, wherein the synthetic composition comprises a least 1E+03 endophytes per seed. 25. The synthetic composition of Claim 21, wherein the synthetic composition comprises a least 1E+04 endophytes per seed. 26. The synthetic composition of Claim 20, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 27. The synthetic composition of Claim 26, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, yield, plant height, stand count, and or root weight. 28. The synthetic composition of Claim 27, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 29. The synthetic composition of Claim 28, wherein the one or more pests or pathogens is a Pythium, or Fusarium species. 30. The synthetic composition of Claim 26, wherein: the synthetic composition additionally comprises one or more soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species. 31. The synthetic composition of Claim 26, wherein: the synthetic composition additionally comprises one or more wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 32. The synthetic composition of Claim 26, wherein: the synthetic composition additionally comprises one or more cotton plant elements, the one or more traits of agronomic importance are shoot weight, root weight, plant height, stand count, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 33. The synthetic composition of Claim 26, wherein: the synthetic composition additionally comprises one or more wheat plant elements, the one or more traits of agronomic importance are yield, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species, Rhizoctonia species, and Fusarium species. 34. The synthetic composition of Claim 19, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.132. 35. The synthetic composition of Claim 19, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs.133, 134, 135, and 136. 36. The synthetic composition of Claim 19, wherein the one or more endophytes are of the genus Bacillus. 37. The synthetic composition of Claim 19, wherein the one or more endophytes are of the genus and species Bacillus velenzensis. 38. The synthetic composition of Claim 19, wherein the solid carrier is talc and the adherent is mineral oil. 39. A synthetic composition, comprising one or more endophytes heterologously disposed to a treatment formulation, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO.27, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.28-31. 40. The synthetic composition of Claim 39, wherein the synthetic composition additionally comprises one or more plant element elements. 41. The synthetic composition of Claim 40, wherein the one or more plant element elements are seeds. 42. The synthetic composition of Claim 40, wherein the one or more plant element elements are soybean or wheat. 43. The synthetic composition of Claim 41, wherein the synthetic composition comprises a least 1E+02 endophytes per seed. 44. The synthetic composition of Claim 41, wherein the synthetic composition comprises a least 1E+03 endophytes per seed. 45. The synthetic composition of Claim 41, wherein the synthetic composition comprises a least 1E+04 endophytes per seed. 46. The synthetic composition of Claim 40, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 47. The synthetic composition of Claim 46, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance and or shoot fresh weight. 48. The synthetic composition of Claim 47, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 49. The synthetic composition of Claim 48, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 50. The synthetic composition of Claim 46, wherein: the synthetic composition additionally comprises one or more soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium or Fusarium species. 51. The synthetic composition of Claim 46, wherein: the synthetic composition additionally comprises one or more wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium or Rhizoctoina species. 52. The synthetic composition of Claim 39, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.27. 53. The synthetic composition of Claim 39, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs.28, 29, 30, and 31. 54. The synthetic composition of Claim 39, wherein the one or more endophytes are of the genus Pseudomonas. 55. The synthetic composition of Claim 39, wherein the one or more endophytes are of the genus and species Pseudomonas glycinis. 56. The synthetic composition of Claim 39, wherein the solid carrier is talc and the adherent is mineral oil. 57. A synthetic composition, comprising one or more endophytes heterologously disposed to a treatment formulation, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO.32 or SEQ ID NOs. 59-94, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.95-127. 58. The synthetic composition of Claim 57, wherein the synthetic composition additionally comprises one or more plant element elements. 59. The synthetic composition of Claim 58, wherein the one or more plant element elements are seeds. 60. The synthetic composition of Claim 58, wherein the one or more plant element elements are soybean, cotton, or wheat. 61. The synthetic composition of Claim 59, wherein the synthetic composition comprises a least 1E+02 endophytes per seed. 62. The synthetic composition of Claim 59, wherein the synthetic composition comprises a least 1E+03 endophytes per seed. 63. The synthetic composition of Claim 59, wherein the synthetic composition comprises a least 1E+04 endophytes per seed. 64. The synthetic composition of Claim 58, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 65. The synthetic composition of Claim 64, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, root weight, shoot weight, yield, early emergence, full emergence, and or plant height. 66. The synthetic composition of Claim 65, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 67. The synthetic composition of Claim 66, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 68. The synthetic composition of Claim 64, wherein: the synthetic composition additionally comprises one or more soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight, root weight, shoot weight, yield, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species, a Rhizoctonia species, or Fusarium species. 69. The synthetic composition of Claim 64, wherein: the synthetic composition additionally comprises one or more wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium or Rhizoctoina species. 70. The synthetic composition of Claim 57, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.32 or SEQ ID NOs.59-94. 71. The synthetic composition of Claim 57, wherein the one or more endophytes are capable of one or more producing proteins having amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.95-127. 72. The synthetic composition of Claim 57, wherein the genome of the one or more endophytes comprises at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.61, 71, 72, 75, 76, 80, 81, 82, 85, 87, 88, and 89, and at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.59, 60, 63, 64, 65, and 84, and at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.67, 70, 79, and 83, and at least a polynucleotide region having at least 97% sequence identity to SEQ ID NO. 77, and at least one polynucleotide regions having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.62, 66, 68, 69, 73, 74, 78, and 86. 73. The synthetic composition of Claim 57, wherein the genome of the one or more endophytes comprises at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs. 61, 71, 72, 75, 76, 80, 81, 82, 85, 87, 88, and 89, and at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.59, 60, 63, 64, 65, and 84, and at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.67, 70, 79, and 83, and at least a polynucleotide region having 100% sequence identity to SEQ ID NO.77, and at least one polynucleotide regions having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.62, 66, 68, 69, 73, 74, 78, and 86. 74. The synthetic composition of Claim 57, wherein the one or more endophytes are of the genus Trichoderma. 75. The synthetic composition of Claim 57, wherein the one or more endophytes are of the genus and species Trichoderma hamatum. 76. The synthetic composition of Claim 57, wherein the treatment formulation comprises a solid carrier and adherent. 77. The synthetic composition of Claim 57, wherein the solid carrier is talc and the adherent is mineral oil. 78. A method of improving plant health, comprising applying a one or more heterologously disposed endophytes to a plant element, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO. 127, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.128-131. 79. The method of Claim 78, wherein the one or more plant element elements are soybean, wheat, or corn. 80. The method of Claim 78, wherein the one or more plant element elements are seeds. 81. The method of Claim 80, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+02 endophytes per seed. 82. The method of Claim 80, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+03 endophytes per seed. 83. The method of Claim 80, wherein the one or more heterologously disposed endophytes are present in an average abundance of 1E+04 endophytes per seed. 84. The method of Claim 78, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 85. The method of Claim 84, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, yield, plant height, shoot weight, and or root weight. 86. The method of Claim 85, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 87. The method of Claim 86, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 88. The synthetic composition of Claim 84, wherein: the one or more plant elements are soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium, Rhizoctonia, or Fusarium species. 89. The method of Claim 84, wherein: the one or more plant elements are wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 90. The method of Claim 84, wherein: the one or more plant elements are corn plant elements, the one or more traits of agronomic importance are shoot weight, yield, plant height, root weight, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 91. The method of Claim 78, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.127. 92. The method of Claim 78, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs. 128, 129, 130, and 131. 93. The method of Claim 78, wherein the one or more endophytes are of the genus Chitinophaga. 94. The method of Claim 78, wherein the one or more endophytes are of the genus and species Chitinophaga oryzae. 95. A method of improving plant health, comprising applying a one or more heterologously disposed endophytes to a plant element, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO. 132, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.133-136. 96. The method of Claim 95, wherein the one or more plant element elements are seeds. 97. The method of Claim 95, wherein the one or more plant element elements are soybean, wheat, or cotton. 98. The method of Claim 95, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+02 endophytes per seed. 99. The method of Claim 95, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+03 endophytes per seed. 100. The method of Claim 95, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+04 endophytes per seed. 101. The method of Claim 95, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 102. The method of Claim 101, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, yield, plant height, stand count, and or root weight. 103. The method of Claim 102, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 104. The method of Claim 103, wherein the one or more pests or pathogens is a Pythium, or Fusarium species. 105. The method of Claim 101, wherein: the one or more plant elements are soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species. 106. The method of Claim 101, wherein: the one or more plant elements are wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 107. The method of Claim 101, wherein: the one or more plant elements are cotton plant elements, the one or more traits of agronomic importance are shoot weight, root weight, plant height, stand count, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium species. 108. The method of Claim 101, wherein: the one or more plant elements are wheat plant elements, the one or more traits of agronomic importance are yield, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species, Rhizoctonia species, and Fusarium species. 109. The method of Claim 95, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.132. 110. The method of Claim 95, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs. 133, 134, 135, and 136. 111. The method of Claim 95, wherein the one or more endophytes are of the genus Bacillus. 112. The method of Claim 95, wherein the one or more endophytes are of the genus and species Bacillus velenzensis. 113. The method of Claim 95, wherein the one or more endophytes are applied in a treatment formulation comprising a solid carrier and an adherent. 114. The method of Claim 113, wherein the solid carrier is talc and the adherent is mineral oil. 115. A method of improving plant health, comprising applying a one or more heterologously disposed endophytes to a plant element, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO. 27, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.28-31. 116. The method of Claim 115, wherein the one or more plant element elements are seeds. 117. The method of Claim 115, wherein the one or more plant element elements are soybean or wheat. 118. The method of Claim 115, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+02 endophytes per seed. 119. The method of Claim 115, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+03 endophytes per seed. 120. The method of Claim 115, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+04 endophytes per seed. 121. The method of Claim 115, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 122. The method of Claim 121, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance and or shoot fresh weight. 123. The method of Claim 122, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 124. The method of Claim 123, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 125. The method of Claim 121, wherein: the one or more plant elements are soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium or Fusarium species. 126. The method of Claim 121, wherein: the one or more plant elements are wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium or Rhizoctoina species. 127. The method of Claim 115, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.27. 128. The method of Claim 115, wherein the one or more endophytes are capable of producing proteins having amino acid sequences are at least 97% identical to SEQ ID NOs. 28, 29, 30, and 31. 129. The method of Claim 115, wherein the one or more endophytes are of the genus Pseudomonas. 130. The method of Claim 115, wherein the one or more endophytes are of the genus and species Pseudomonas glycinis. 131. The method of Claim 115, wherein the one or more endophytes are applied in a treatment formulation comprising a solid carrier and an adherent. 132. The method of Claim 115, wherein the solid carrier is talc and the adherent is mineral oil. 133. A method of improving plant health, comprising applying a one or more heterologously disposed endophytes to a plant element, wherein the one or more endophytes comprise at least one polynucleotide sequence that is at least 97% identical to SEQ ID NO. 32 or SEQ ID NOs.59-94, and the genomes of the one or more endophytes comprise one or more open reading frames encoding proteins whose amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.95-127. 134. The method of Claim 133, wherein the one or more plant element elements are seeds. 135. The method of Claim 133, wherein the one or more plant element elements are soybean or wheat. 136. The method of Claim 134, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+02 endophytes per seed. 137. The method of Claim 134, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+03 endophytes per seed. 138. The method of Claim 134, wherein the one or more heterologously disposed endophytes are present in an average abundance of a least 1E+04 endophytes per seed. 139. The method of Claim 133, wherein the one or more endophytes are capable of improving one or more traits of agronomic importance in a plant derived from the plant element relative to a plant derived from a reference plant element. 140. The method of Claim 139, wherein the one or more traits of agronomic importance comprise one or more of biotic stress tolerance, shoot fresh weight, root weight, yield, early emergence, full emergence, and or plant height. 141. The method of Claim 140, wherein the biotic stress is a growth environment comprising one or more pests or pathogens. 142. The method of Claim 141, wherein the one or more pests or pathogens is a Pythium, Rhizoctonia, or Fusarium species. 143. The method of Claim 139, wherein: the one or more plant elements are soybean plant elements, the one or more traits of agronomic importance are shoot fresh weight, root weight, shoot weight, yield, and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Pythium species, a Rhizoctonia species, or Fusarium species. 144. The method of Claim 139, wherein: the one or more plant elements are wheat plant elements, the one or more traits of agronomic importance are shoot fresh weight and biotic stress tolerance, wherein biotic stress is a growth environment comprising a Fusarium or Rhizoctoina species. 145. The method of Claim 133, wherein the one or more endophytes comprise at least one polynucleotide sequence that is 100% identical to SEQ ID NO.32 or SEQ ID NOs.59- 94. 146. The method of Claim 133, wherein the one or more endophytes are capable of one or more producing proteins having amino acid sequences are at least 97% identical to one or more of SEQ ID NOs.95-127. 147. The method of Claim 133, wherein the genomes of the one or more endophytes comprises at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.61, 71, 72, 75, 76, 80, 81, 82, 85, 87, 88, and 89, and at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.59, 60, 63, 64, 65, and 84, and at least one polynucleotide region having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.67, 70, 79, and 83, and at least a polynucleotide region having at least 97% sequence identity to SEQ ID NO.77, and at least one polynucleotide regions having at least 97% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.62, 66, 68, 69, 73, 74, 78, and 86. 148. The method of Claim 133, wherein the genome of the one or more endophytes comprises at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.61, 71, 72, 75, 76, 80, 81, 82, 85, 87, 88, and 89, and at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.59, 60, 63, 64, 65, and 84, and at least one polynucleotide region having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.67, 70, 79, and 83, and at least a polynucleotide region having 100% sequence identity to SEQ ID NO.77, and at least one polynucleotide regions having 100% sequence identity to one or more polynucleotide sequences selected from the group consisting of SEQ ID NOs.62, 66, 68, 69, 73, 74, 78, and 86. 149. The method of Claim 133, wherein the one or more endophytes are of the genus Trichoderma. 150. The method of Claim 133, wherein the one or more endophytes are of the genus and species Trichoderma hamatum. 151. The method of Claim 133, wherein the one or more endophytes are applied in a treatment formulation comprising a solid carrier and an adherent. 152. The method of Claim 151, wherein the solid carrier is talc and the adherent is mineral oil. |
Table 1. Sources of microbes of the present invention [099] Each sample was processed independently. Each sample was washed in a dilute water and detergent solution; tissue was collected from plants. Samples were surface sterilized by successive rinses: 2 minutes in 10% bleach solution, 2 minutes in 70% ethanol solution, and a rinse with sterile water. The series of rinses was repeated 3 times. The plant tissue was cut into small pieces with sterile scissors and blended with 3, 7 mm steel beads in 5-7.5 ml phosphate buffered solution (PBS). DNA was extracted from the ground tissues using the Magbind Plant DNA kit (Omega, Norcross, Georgia, USA) according to the manufacturer’s instructions. Identification of endophytes by sequencing of marker genes [0100] The endophytes were characterized by the sequences of genomic regions. Primers that amplify genomic regions of the endophytes of the present invention are listed in Table 2. Sanger sequencing was performed at Genewiz (South Plainfield, NJ). Raw chromatograms were converted to sequences, and corresponding quality scores were assigned using TraceTuner v3.0.6beta (US 6,681,186). These sequences were quality filtered, aligned and a consensus sequence generated using Geneious v 8.1.8 (Biomatters Limited, Auckland NZ). The consensus sequences identifying the endophytes are listed in Table 3. Table 2. Primer sequences useful in identifying microbes of the present invention
Table 3. Exemplary sequences of endophytes of the present invention
Example 2. Taxonomic classification of endophytes [0101] Classification of strains was done by the following methodology. [0102] To accurately characterize isolated bacterial endophytes, colonies were submitted for marker gene sequencing, and the sequences were analyzed to provide taxonomic classifications. Colonies were subjected to 16S rRNA gene PCR amplification using a primer pair 27f (5’- AGAGTTTGATYMTGGCTCAG-3’) (SEQ ID NO: 1) and 1492r (5’- GGTTACCTTGTTACGACTT-3’) (SEQ ID NO: 2). Sequencing reactions were performed using primers: 27f (5’- AGAGTTTGATYMTGGCTCAG -3’) (SEQ ID NO: 1), 515f (5’ – GTGYCAGCMGCCGCGGTAA- 3’) (SEQ ID NO: 3), 806r (5’- GGACTACNVGGGTWTCTAAT- 3’) (SEQ ID NO: 4), and 1492r (5’- GGTTACCTTGTTACGACTT -3’) (SEQ ID NO: 2). To accurately characterize isolated fungal endophytes, genomic DNA isolated as above was submitted for marker gene sequencing, and the sequences were analyzed to provide taxonomic classifications. PCR was used to amplify the nuclear ribosomal internal transcribed spacers (ITS) region using the primer pair ITS_1 (5’- CTTGGTCATTTAGAGGAAGTAA -3’) (SEQ ID NO: 5) and LR5 (5’- TCCTGAGGGAAACTTCG -3’) (SEQ ID NO: 6). Each 25 microliter-reaction mixture included 22.5 microliters of Invitrogen Platinum Taq supermix, 0.5 microliter of each primer (10 micromolar), and 1.5 microliters of DNA template (~2-4 ng). Cycling reactions were run with MJ Research PTC thermocyclers and consisted of 94°C for 5 min, 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min, and 72°C for 10 min. Sanger sequencing of was performed at Genewiz (South Plainfield, NJ) using primers: ITS_1 (5’- CTTGGTCATTTAGAGGAAGTAA -3’) (SEQ ID NO: 5), ITS_2 (5’- GCTGCGTTCTTCATCGATGC -3’) (SEQ ID NO: 7), ITS_3 (5’- GCATCGATGAAGAACGCAGC-3’) (SEQ ID NO: 8), and LR5 (5’- TCCTGAGGGAAACTTCG -3’) (SEQ ID NO: 6). Preferably sequencing primers were chosen so that overlapping regions are sequenced. Sanger sequencing of marker genes was performed at Genewiz (South Plainfield, NJ). Raw chromatograms were converted to sequences, and corresponding quality scores were assigned using TraceTuner v3.0.6beta (US 6,681,186). These sequences were quality filtered, aligned and a consensus sequence generated using Geneious v 8.1.8 (Biomatters Limited, Auckland NZ). [0103] Taxonomic classifications were assigned to the sequences using the highest probability of assignment based on the results of industry standard taxonomic classification tools: LCA (runs USEARCH (Edgar, R. C., 2010) with option search_global, then for all best match hits, returns lowest taxonomic rank shared by all best hits for a query), RDP Naive Bayesian rRNA Classifier version 2.11, September 2015 (Wang et al., 2007), SPINGO version 1.3 (32 bit) (Allard et al. (2015) BMC Bioinformatics 16:324 DOI: 10.1186/s12859- 015-0747-1), and UTAX version v8.1.1861_i86linux64 (Edgar, R.C. (2016) available online at drive5.com/usearch/manual/utax_algo.html), using reference databases: RDP 16S rRNA training set 15 (Cole et al. (2014) Nucleic Acid Research, 42 (Database issue): D633-D642), and SILVA version 119 (Quast et al. (2013) Nucleic Acid Research, 41 (Database issue): D590-D596). The classifier and database combinations listed in Table 4 were used to assign taxonomy to bacterial sequences. Table 4. The classifier and database combinations used to classify 16S rRNA gene or ITS sequences Table 5. Taxonomic classification of endophytes of the present invention Example 3. Assessment of improved plant characteristics: Vigor assay Assay of soy seedling vigor [0104] Seed preparation: The lot quality of soybean seeds is first assessed by testing germination of 100 seeds. Seeds are placed, 8 seeds per petri dish, on filter paper in petri dishes, 12 ml of water is added to each plate and plates are incubated for 3 days at 24°C. The process should be repeated with a fresh seed lot if fewer than 95% of the seeds have germinated. One thousand soybean seeds are then surface sterilized by co-incubation with chlorine gas in a 20 x 30 cm container placed in a chemical fume hood for 16 hours. Percent germination of 50 seeds, per sterilization batch, is tested as above and confirmed to be greater than 95%. [0105] Preparation of endophyte treatments: Spore solutions are made by rinsing and scraping spores from agar slants which have been growing for about 1 month. Rinsing is done with 0.05% Silwet. Solutions are passed through Miracloth to filter out mycelia. Spores per ml are counted under a microscope using a hemocytometer. The stock suspension is then diluted into 10^6 spores/ml utilizing water.3 µl of spore suspension is used per soy seed (~10^3 CFUs/seed is obtained). Control treatments are prepared by adding equivalent volumes of sterile water to seeds. [0106] Assay of seedling vigor: Two rolled pieces of germination paper are placed in a sterile glass gar with 50 ml sterile water, then removed when completely saturated. Then the papers are separated, and inoculated seeds are placed at approximately 1 cm intervals along the length of one sheet of moistened germination paper, at least 2.5 cm from the top of the paper and 3.8 cm from the edge of the paper. The second sheet of is placed on top of the soy seeds and the layered papers and seeds are loosely rolled into a tube. Each tube is secured with a rubber band around the middle and placed in a single sterile glass jar and covered loosely with a lid. For each treatment, three jars with 15 seeds per jar are prepared. The position of jars within the growth chamber is randomized. Jars are incubated at 60% relative humidity, and 22°C day, 18°C night with 12 hours light and 12 hours dark for 4 days and then the lids are removed, and the jars incubated for an additional 7 days. Then the germinated soy seedlings are weighed and photographed, and root length and root surface area are measured. [0107] Dirt, excess water, seed coats and other debris is removed from seedlings to allow accurate scanning of the roots. Individual seedlings are laid out on clear plastic trays and trays are arranged on an Epson Expression 11000XL scanner (Epson America, Inc., Long Beach CA). Roots are manually arranged to reduce the amount of overlap. For root measurements, shoots are removed if the shape of the shoot causes it to overlap the roots. [0108] The WinRHIZO software version Arabidopsis Pro2016a (Regents Instruments, Quebec Canada) is used with the following acquisition settings: greyscale 4000 dpi image, speed priority, overlapping (1 object), Root Morphology: Precision (standard), Crossing Detection (normal). The scanning area is set to the maximum scanner area. When the scan is completed, the root area is selected, and root length and root surface area are measured. [0109] Statistical analysis is performed using R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/) or a similar statistical software program. Assay of rice seedling vigor [0110] Seed preparation: The lot of rice seeds is first evaluated for germination by transfer of 100 seeds and with 8 ml of water to a filter paper lined petri dish. Seeds are incubated for 3 days at 24°C. The process should be repeated with a fresh seed lot if fewer than 95% of the seeds have germinated. Rice seeds are then surface sterilized by co-incubation with chlorine gas in a 20 x 30 cm container in a chemical fume hood for 12 hours. Percent germination of 50 seeds, per sterilization batch, is tested as above and confirmed to be greater than 95%. [0111] Optional reagent preparation: 7.5% polyethylene glycol (PEG) is prepared by adding 75 g of PEG to 1000 ml of water, then stirring on a warm hot plate until the PEG is fully dissolved. The solution is then autoclaved. [0112] Preparation of endophyte treatments: Spore solutions are made by rinsing and scraping spores from agar slants which have been growing for about 1 month. Rinsing was done with 0.05% Silwet. Solutions are passed through Miracloth to filter out mycelia. Spores per ml are counted under a microscope using a hemocytometer. The stock suspension is then diluted into 10^6 spores/ml utilizing water.3 µl of spore suspension is used per rice seed (~10^3 CFUs/seed was obtained). Seeds and spores are combined in a 50 ml falcon tube and gently shaken for 5-10 seconds until thoroughly coated. Control treatments are prepared by adding equivalent volumes of sterile water to seeds. [0113] Assay of seedling vigor: Petri dishes are prepared by adding four sheets of sterile heavy weight seed germination paper, then adding either 50 ml of sterile water or, optionally, 50 ml of PEG solution as prepared above, to each plate then allowing the liquid to thoroughly soak into all sheets. The sheets are positioned and then creased so that the back of the plate and one side wall are covered, two sheets are then removed and placed on a sterile surface. Along the edge of the plate across from the covered side wall 15 inoculated rice seeds are placed evenly at least one inch from the top of the plate and half an inch from the sides. Seeds are placed smooth side up and with the pointed end of the seed pointing toward the side wall of the plate covered by germination paper. The seeds are then covered by the two reserved sheets, and the moist paper layers smoothed together to remove air bubbles and secure the seeds, and then the lid is replaced. For each treatment, at least three plates with 15 seeds per plate are prepared. The plates are then randomly distributed into stacks of 8-12 plates and a plate without seeds is placed on the top. The stacks are incubated at 60% relative humidity, and 22°C day, 18°C night with 12 hours light and 12 hours dark for 24 hours, then each plate is turned to a semi-vertical position with the side wall covered by paper at the bottom. The plates are incubated for an additional 5 days, then rice seeds are scored manually for germination, root and shoot length. [0114] Statistical analysis is performed using R or a similar statistical software program. Assay of corn seedling vigor [0115] Seed preparation: The lot quality of corn seeds is first evaluated for germination by transfer of 100 seeds with 3.5 ml of water to a filter paper lined petri dish. Seeds are incubated for 3 days at 24°C. The process should be repeated with a fresh seed lot if fewer than 95% of the seeds have germinated. One thousand corn seeds are then surface sterilized by co-incubation with chlorine gas in a 20 x 30 cm container in a chemical fume hood for 12 hours. Percent germination of 50 seeds, per sterilization batch, is tested as above and confirmed to be greater than 95%. [0116] Optional reagent preparation: 7.5% PEG 6000 (Calbiochem, San Diego, CA) is prepared by adding 75 g of PEG to 1000 ml of water, then stirred on a warm hot plate until the PEG is fully dissolved. The solution is then autoclaved. [0117] Preparation of endophyte treatments: Spore solutions are made by rinsing and scraping spores from agar slants which have been growing for about 1 month. Rinsing is done with 0.05% Silwet. Solutions are passed through Miracloth to filter out mycelia. Spores per ml are counted under a microscope using a hemocytometer. The stock suspension is then diluted into 10^6 spores/ml utilizing water.3 µl of spore suspension is used per corn seed (~10^3 CFUs/seed is obtained). Control treatments are prepared by adding equivalent volumes of sterile water to seeds. [0118] Assay of seedling vigor: Either 25 ml of sterile water or, optionally, 25 ml of PEG solution as prepared above, is added to each CygTM germination pouch (Mega International, Newport, MN) and place into pouch rack (Mega International, Newport, MN). Sterile forceps are used to place corn seeds prepared as above into every other perforation in the germination pouch. Seeds are fitted snugly into each perforation to ensure they do not shift when moving the pouches. Before and in between treatments forceps are sterilized using ethanol and flame and workspace wiped down with 70% ethanol. For each treatment, three pouches with 15 seeds per pouch are prepared. The germination racks with germination pouches are placed into plastic tubs and covered with perforated plastic wrap to prevent drying. Tubs are incubated at 60% relative humidity, and 22°C day, 18°C night with 12 hours light and 12 hours dark for 6 days to allow for germination and root length growth. Placement of pouches within racks and racks/tubs within the growth chamber is randomized to minimize positional effect. At the end of 6 days the corn seeds are scored manually for germination, root and shoot length. [0119] Statistical analysis is performed using R or a similar statistical software program. Assay of wheat seedling vigor [0120] Seed preparation: The lot of wheat seeds is first evaluated for germination by transfer of 100 seeds and with 8 ml of water to a filter paper lined petri dish. Seeds are incubated for 3 days at 24°C. The process should be repeated with a fresh seed lot if fewer than 95% of the seeds have germinated. Wheat seeds are then surface sterilized by co-incubation with chlorine gas in a 20 x 30 cm container in a chemical fume hood for 12 hours. Percent germination of 50 seeds, per sterilization batch, is tested as above and confirmed to be greater than 95%. [0121] Optional reagent preparation: 7.5% polyethylene glycol (PEG) is prepared by adding 75 g of PEG to 1000 ml of water, then stirring on a warm hot plate until the PEG is fully dissolved. The solution is then autoclaved. [0122] Preparation of endophyte treatments: Spore solutions are made by rinsing and scraping spores from agar slants which have been growing for about 1 month. Rinsing is done with 0.05% Silwet. Solutions are passed through Miracloth to filter out mycelia. Spores per ml are counted under a microscope using a hemocytometer. The stock suspension is then diluted into 10^6 spores/ml utilizing water.3 µl of spore suspension is used per wheat seed (~10^3 CFUs/seed was obtained). Seeds and spores are combined a 50 ml falcon tube and gently shaken for 5-10 seconds until thoroughly coated. Control treatments are prepared by adding equivalent volumes of sterile water to seeds. [0123] Assay of seedling vigor: Petri dishes are prepared by adding four sheets of sterile heavy weight seed germination paper, then adding either 50 ml of sterile water or, optionally, 50 ml of PEG solution as prepared above, to each plate then allowing the liquid to thoroughly soak into all sheets. The sheets are positioned and then creased so that the back of the plate and one side wall are covered, two sheets are then removed and placed on a sterile surface. Along the edge of the plate across from the covered side wall 15 inoculated wheat seeds are placed evenly at least one inch from the top of the plate and half an inch from the sides. Seeds are placed smooth side up and with the pointed end of the seed pointing toward the side wall of the plate covered by germination paper. The seeds are then covered by the two reserved sheets, and the moist paper layers smoothed together to remove air bubbles and secure the seeds, and then the lid is replaced. For each treatment, at least three plates with 15 seeds per plate are prepared. The plates are then randomly distributed into stacks of 8-12 plates and a plate without seeds is placed on the top. The stacks are incubated at 60% relative humidity, and 22°C day, 18°C night with 12 hours light and 12 hours dark for 24 hours, then each plate is turned to a semi-vertical position with the side wall covered by paper at the bottom. The plates are incubated for an additional 5 days, then wheat seeds are scored manually for scored manually for germination, root and shoot length, root and shoot surface area, seedling mass, root and shoot and seedling length. [0124] Statistical analysis is performed using R or a similar statistical software program. Example 4. Method of preparation of endophytes and heterologous disposition of endophytes on seeds [0125] Seeds are heterologously disposed to each endophyte according to the following seed treatment protocol. Preparation of seeds [0126] The average weight of seeds is calculated by weighing 3 samples of 100 size selected seeds each and calculating the average weight of a seed. This value is used to calculate the target dose of endophyte per seed. The target dose is generally between 10^0 - 10^6 CFU per seed, in some cases at least 10^3 CFU per seed, or at least 10^4 CFU per seed. Table Z lists the target dose for endophytes of the present invention as applied to larger seeds (for example corn, soy and cotton) and smaller seeds (for example, wheat, rice, barley, oats). Other types of plants may be treated, and dosage determined based on the similarity of those seeds to the seeds listed in Table Z. Table Z. Target doses in for endophytes of the present invention by crop type. Preparation of bacterial and fungal endophytes [0127] MIC-54347 is produced by soild state fermentation. A seed flask containing potato dextrose broth (PDB) is inoculated with MIC-54347 and incubated for 7 days. Soil substrate consisting of 33% millet, 9.4% vermiculite, 9.4% clay, 2.8% wheat bran, 0.6% yeast extract, 45% water is inoculated with the seed culture, and the culture grown for approximately 14 days at 24 C. Total biomass is collected. The total volume of inoculum needed to treat the seeds with the desired dose was calculated based on the target dose. The target dose is generally between 10^0 - 10^6 CFU per seed, in some cases at least 10^3 CFU per seed, or at least 10^4 CFU per seed. The inoculum is diluted with sterile 1x PBS so that the total volume of inoculum per seed is about 2.5 ul/seed for corn, about 1.5 microliters/seed for wheat and soy, and about 1.5 microliters/seed for cotton. Control treatments were prepared using equivalent volumes of sterile 1x PBS. The inoculum solution is combined with a treatment formulation containing talc and mineral oil and is applied to the prepared seeds and mixed well. [0128] MIC-67967, MIC-84302, and MIC-18905 are produced by liquid state fermentation. A seed flask containing trypticase soy broth (TSB) is inoculated with the endophyte and incubated for 24 hours. Liquid fermentation is completed in a bioreactor. Total biomass is collected. The target dose is generally between 10^0 - 10^6 CFU per seed, in some cases at least 10^3 CFU per seed, or at least 10^4 CFU per seed. The biomass suspension is diluted with sterile 1x PBS so that the total volume of inoculum per seed is about 2.5 ul/seed for corn, about 1.5 ul/seed for wheat and soy, and about 1.5 ul/seed for cotton. Control treatments were prepared using equivalent volumes of sterile 1x PBS. For MIC-67967 and MIC-84302 the diluted biomass suspension is applied to the prepared seeds directly and mixed well. For MIC-67967 and MIC-84302 the biomass suspension is dried and combined with a treatment formulation containing talc and mineral oil and is applied to the prepared seeds and mixed well. Example 5. Greenhouse assessment of improved plant characteristics under water deficit [0129] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising a water deficit. [0130] Greenhouse assay setup: This greenhouse assay is conducted in individual plastic pots, filled with moistened potting soil. This greenhouse assay is conducted using seeds (optionally, chemically treated) coated with one or more endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Seeds are placed onto each pot and lightly covered with potting mix. Replicated pots of each treatment are set up and placed on a greenhouse bench using a random block design. For example, 18 replicates are planted for each treatment and control. Plants are monitored daily for emergence and watered as necessary to maintain a moist but not saturated soil surface (for example, plants are watered with 125 ml Hoagland’s solution (8 mM N) (Hoagland, D.R. and D.I. Arnon.1950. The water culture method for growing plant without soil. California Agri. Exp. Sta. Cir. No.347. University of California Berkley Press, CA., pp: 347.) per pot on every Monday, Wednesday and Friday). [0131] The following growth and vigor metrics are measured for each treatment: percentage emergence at Day 4, 5, 7 (for soybean, winter wheat and cotton) or Day 3, 4, 5 (for corn), leaf count (the number of fully expanded leaves on the main stem) at Days 10, 17 and 24. [0132] At Day 14 after planting, the potting mix in each pot is fully saturated (for example, 150 ml Hoagland’s solution is added to each pot and the soil given time to fully absorb the solution, before an additional 150 ml Hoagland’s solution). On subsequent days plants are observed and assigned a wilt score. Wilt scores range from 1 - 9 and are more fully described in the following table. Table A. Description of phenotypes for each wilt scores [0133] Watering is withheld until 80% of plants have a wilt score of at least 4. Pots are then fully saturated and a normal watering schedule resumed. Additional vigor and growth metrics may be measured during recovery including shoot height, area of chlorosis, turgor pressure of leaves, number of live leaves, etc. After a recovery period, for example 1 week, plants are gently removed from pots, washed with tap water to remove dirt, and photographed. Optionally, plant tissue is collected for nutrient composition analysis. Plants are put into a paper bag and dried in an oven. Optionally, the plant is separated into shoot and root tissue prior to drying. The dry weight of each individual plant, or shoot or root thereof, is recorded. Example 6. Greenhouse assessment of improved plant characteristics under nitrogen deficit [0134] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising a nitrogen deficit. [0135] Greenhouse assay setup: This greenhouse assay is conducted in individual plastic pots, filled with moistened potting soil. This greenhouse assay is conducted using seeds (optionally, chemically treated) coated with one or more endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Seeds are placed onto each pot and lightly covered with potting mix. Replicated pots of each treatment are set up and placed on a greenhouse bench using a random block design. For example, 18 replicates are planted for each treatment and control. Nitrogen deficit is introduced by reducing the Nitrogen in the Hoagland’s solution (3 mM N), which is used to water the plants. Plants are monitored daily for emergence and watered as necessary to maintain a moist but not saturated soil surface (for example, plants are watered with 125 ml Hoagland’s solution (3 mM N) per pot on every Monday, Wednesday and Friday). [0136] The following growth and vigor metrics are collected for each treatment: percentage emergence at Day 4, 5, 7 (for soybean, winter wheat and cotton) or Day 3, 4, 5 (for corn), leaf count (the number of fully expanded leaves on the main stem) at Days 10, 17 and 24. [0137] Additional vigor and growth metrics may be collected including shoot height, leaf area, number of chlorotic leaves, chlorophyll content, number of live leaves, etc. At harvest plants are gently removed from pots, washed with tap water to remove dirt, and photographed. Plant tissue is collected for nutrient composition analysis. Plants are put into a paper bag and dried in an oven. Optionally, the plant is separated into shoot and root tissue prior to drying. The dry weight of each individual plant, or shoot or root thereof, is recorded. Example 7. Greenhouse assessment of improved plant characteristics under phosphorus deficit [0138] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising a phosphorus deficit. [0139] This greenhouse assay is conducted in individual plastic pots, filled with moistened potting soil. This greenhouse assay is conducted using seeds (optionally, chemically treated) coated with one or more endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Seeds are placed onto each pot and lightly covered with potting mix. Replicated pots of each treatment are set up and placed on a greenhouse bench using a random block design. For example, 16 replicates are planted for each treatment and control. Phosphorus deficit is introduced by removing Phosphorus from the Hoagland’s solution (0 mM P), which is used to water the plants. Plants are monitored daily for emergence and watered as necessary to maintain a moist but not saturated soil surface (for example, plants are watered with 125 ml Hoagland’s solution (0 mM P) per pot on every Monday, Wednesday and Friday). [0140] The following growth and vigor metrics are collected for each treatment: percentage emergence at Day 4, 5, 7 (for soybean, winter wheat and cotton) or Day 3, 4, 5 (for corn), leaf count (the number of fully expanded leaves on the main stem) at Days 10, 17 and 24. [0141] Additional vigor and growth metrics may be collected including shoot height, leaf area, coloration of leaves, number of live leaves, etc. At harvest plants are gently removed from pots, washed with tap water to remove dirt, and photographed. Plant tissue is collected for nutrient composition analysis. Plants are put into a paper bag and dried in an oven. Optionally, the plant is separated into shoot and root tissue prior to drying. The dry weight of each individual plant, or shoot or root thereof, is recorded. Example 8. Greenhouse assessment of improved plant health under biotic stress [0142] This example describes an exemplary method by which improved plant health of endophyte treated plants was shown in a growth environment comprising the crop pathogen Rhizoctonia solani or Pythium ultimum, causal agents of seedling damping off disease. This assay may utilize dicots or monocots, though results for soybean, cotton and wheat are described here. [0143] Preparation of pathogen inoculum A stock of Rhizoctonia solani anastomosis group 4 or Pythium ultimum var. ultimum was grown on a standard potato dextrose agar plate. Plugs of fresh mycelium were then transferred into standard potato dextrose broth. After sufficient growth was achieved, the culture was poured though cheesecloth to capture the fungal biomass, which was subsequently rinsed with water. After removing excess rinse water, a roughly equivalent volume of water was added to the fungal biomass before blending to create a slurry. The resulting slurry was further diluted to the required concentration necessary to observe desired level of symptoms. [0144] Greenhouse assay setup The greenhouse assay was conducted in a commercial potting mix. A divot was placed in the center of a pot containing wetted soil using a standardized dibble. An appropriate volume of slurry was added to the center of each divot. [0145] This greenhouse assay was conducted using seeds coated with one or more endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Seeds were placed onto each divot after addition of the inoculum. The seeds were then covered with uninoculated soil and again watered. High soil moisture levels were maintained throughout the course of the experiment. Replicates were included in a randomized design to obtain sufficient statistical power for analysis. Plants were grown in a controlled environment until approximately 4 days post emergence of control plants. At this point fresh shoot weight was measured on a per plant basis. The assay was repeated multiple times for some endophytes, results are shown in Tables 6 and 7. Table 6. Greenhouse screening of endophytes with activity against Pythium, each line in the table represents an experiment
Table 7. Greenhouse screening of endophytes with activity against Rhizoctonia, each line in the table represents an experiment Example 9. Soybean cyst nematode preparation [0146] The eggs of Heterodera glycines are extracted from soybean stock culture and are used as inoculum for in vitro, growth chamber, greenhouse, and microplot experiments. [0147] In one embodiment, the following method is used. Eggs are extracted from a 60-day- old soybean stock culture maintained in, e.g., 500 ml polystyrene pots. The soil is gently washed from the soybean roots and cysts and females are dislodged from the roots. Water with the cyst and female suspension is poured through nested 850-μm-pore and 250-μm-pore sieves to separate trash from cysts and females. Cysts and females are ground with a mortar and pestle to release the eggs. Eggs are washed with water, collected on a 25-μm-pore sieve, transferred to two 50 ml centrifuge tubes, and spun for 5 minutes at 1,750 r.p.m. The supernatant liquid is then poured off and a sugar solution added (1 lb. cane sugar, l liter water), thoroughly mixing sugar solution and sediment. The suspension is centrifuged at 240 g for 1 minute. The supernatant containing the nematodes is poured on to the 25-μm-pore sieve. After rinsing the sugar away with water, the nematodes are ready for use. For in vitro tests, H. glycines eggs are placed in a modified Baermann funnel (Castillo JD., Lawrence KS., Kloepper JW. Biocontrol of the reniform nematode by Bacillus firmus GB126 and Paecilomyces lilacinus 251 on cotton. Plant Disease.2013; 97: 967–976.) on a Slide Warmer (Model 77) (Marshall Scientific, Brentwood, NH) and incubated at 31°C for 5 to 7 days to obtain the J2. The J2 are collected on a 25-μm-pore sieve, transferred to 1.5 ml microcentrifuge tubes, centrifuged at 5,000 g for 1 minute, rinsed with sterile distilled water, and centrifuged at 5,000 g for 1 minute. The J2 suspensions are adjusted to 30 to 40 J2 per 10 μl of water. Eggs are enumerated at 40× magnification with an inverted TS100 Nikon microscope and standardized to 2,000 eggs per 500 ml polystyrene pot. Example 10. Greenhouse assessment of improved plant health under biotic stress (soybean cyst nematode) [0148] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising the crop pest soybean cyst nematode (Heterodera glycines). [0149] Greenhouse assays are conducted using soybean seeds (optionally, chemically treated soybean seeds) coated with one or more of the endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Microbe treated soybean seeds are planted, infected with nematodes, maintained, and phenotyped in grow rooms. [0150] In one embodiment, the following method is used.98 cones are placed in each cone- tainer to obtain the needed number of cone-tainers. Masks are placed over cones and cones are filled with soil. The cone-tainer is place in a deep pan and water is added until the soil in the cones is saturated. Two soybean seeds are planted 2.5 cm deep in each cone-tainer. Each cone-tainer is placed in a growth tub and watered. [0151] One ml containing 2,000 H. glycines eggs is pipetted into each cone-tainer at planting or the desired number of days after planting. Seedlings are thinned to one per cone-tainer after emergence and watered as appropriate. [0152] Phenotyping is performed as follows. The height of each plant is measured, e.g., by placing the ruler on the lip of a cell and measuring the plant’s height to the nearest millimeter. The mass of each plant is measured, e.g., by cutting the plant at the soil surface, placing the shoot in the weighing container, allowing the weight to stabilize, and autorecording the mass via the scale’s software. The number of H. glycines cysts may be counted after extraction from soybean roots as described herein. The water suspension containing 150 cm^3 of soil is poured through nested 75-μm and 25-μm-pore sieves to extract vermiform stages (juveniles and males). Vermiform stages are collected on the 75-μm-pore sieve and centrifuged using, e.g., the sucrose centrifugation-flotation method. Example 11. Greenhouse assessment of improved plant health under biotic stress (soybean aphid) [0153] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising the crop pest soybean aphid (Aphis glycines). [0154] Greenhouse assays are conducted using soybean seeds (optionally, chemically treated soybean seeds) coated with one or more of the endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Microbe treated soybean seeds are planted, infected with soybean aphids (Aphis glycines), maintained in grow rooms, and phenotyped. [0155] In one embodiment, the following method is used.98 cones are placed in each cone- tainer to obtain the needed number of cone-tainers. Masks are placed over cones and cones are filled with potting medium or soil. The cone-tainer is place in a deep pan and water is added until the soil in the cones is saturated. One soybean seed is planted in each cone-tainer. Each cone-tainer is placed in a growth tub and watered. [0156] A community of soybean aphids is maintained on a stock of soybean plants. To prepare for infestation of the experimental plants, leaves are removed from infested soybean plants from the stock community. One or more leaves are examined under a stereoscope to make sure the aphids are alive and vigorous. Infested leaf cutlets are placed in square plates to maintain leaves alive until the treatment plants are infested with aphids. In some embodiments, 20 infested leaf cutlets are used per each 98 cone tray used in the experiment. The infested leaf cutlets are introduced to the growth environment of the experimental plants at planting or the desired number of days after planting, in some embodiments, 9 days after planting. The experimental cone-tainers are infested following an infestation pattern to allow for aphid choice feeding in planta. The infested experimental plants are maintained in their growth environment until phenotyping. [0157] The plants may be phenotyped at one or more times after infestation, for example 1 day, 4 days, 7 days or more after infestation. Measurement of one or more traits of agronomic importance is performed as follows. The height of each plant is measured, e.g., by placing the ruler on the lip of a cell and measuring the plant’s height to the nearest millimeter or using an automated tool such as a Phenospex PlantEye 3D laser scanner (Phenospex B.V., Heerlen, The Netherlands) . Other traits of agronomic importance may be measured either manually or using a tool such as the Phenospex PlantEye 3D laser scanner, for example the greenness of the plants and the leaf and/or above ground plant area. The mass of each plant may be measured for example via destructive sampling, e.g., by cutting the plant at the soil surface, placing the shoot in the weighing container, allowing the weight to stabilize, and autorecording the mass via the scale’s software. The experimental plants may be maintained through their reproductive stages, and traits of agronomic importance such as number of flowers, number of pods and number of seeds per pod may be measured. Example 12. Greenhouse assessment of improved plant health under biotic stress [0158] This example describes an exemplary method by which improved plant health of endophyte treated plants was shown in a growth environment comprising the crop pathogen Fusarium sp., one of the causal agents of seedling damping off disease. This assay may utilize dicots or monocots, including, for example, soybean and wheat as shown here. [0159] Preparation of Fusarium sp. inoculum A stock of Fusarium sp. was grown on a standard potato dextrose agar plate. Plugs of fresh mycelium were then transferred into breathable bag containing a sterile mixture of water and grain such as sorghum or millet. After sufficient growth is achieved, the culture was removed from the bags and dried. After drying the biomass was coarsely ground. [0160] Greenhouse assay setup The greenhouse assay was conducted in a media mixture consisting of a commercial potting mix and a minimum of 50% inert inorganic material. An appropriate volume of ground pathogen was added to the soil mixture to obtain moderate to severe symptoms. [0161] This greenhouse assay was conducted using seeds coated with one or more endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. A seed was added to the surface of the infested media. The seed was then covered with media lacking pathogen and again watered. High soil moisture levels were maintained throughout the course of the experiment. Replicates were included in a randomized design to obtain sufficient statistical power for analysis. Plants were grown in a controlled environment until approximately 4 days post emergence of control plants. At this point shoot fresh weight was measured on a per plant basis. The assay was repeated multiple times for some endophytes, results are shown in Table 8. Table 8. Greenhouse screening of endophytes with activity against Fusarium, each line in the table represents an experiment
Example 13. Field assessment of improved plant health of soy under biotic stress [0162] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising the crop pests root knot nematode (Meloidogyne incognita), Reniform nematode (Rotylenchulus reniformis), and, opportunistically, the fungal pathogen Fusarium virguliforme. [0163] Field trials are conducted using chemically treated soy seeds coated with one or more of the endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as a flowable powder described in Example 4. Plots for in-field assessment harbor populations of root knot nematode and Reniform nematode, respectively, at an approximately 1.0+E04 eggs per gram of fresh root weight. Opportunistically, these plots are infected with natural inoculum of Fusarium virguliforme, the causal agent of Fusarium Sudden Death Syndrome (SDS). Replicate plots, preferably at least 4 replicate plots, are planted per endophyte or control treatment in a randomized complete block design. Each plot consists of a 7.62 m (25 ft.) by 0.76 m (2.5 ft.) row. The following early growth metrics are measured: percent emergence at 14 days post planting, standing count at 28 and 45 days post planting, plant vigor at 14, 28, and 45 days post planting, plant height at 45 days post planting, fresh shoot weight, fresh root weight, disease rating at a 0-3 scale (3 denotes strong disease symptoms) using the split-root scoring system at 45 days post planting, nematode count at 45 days post planting, and yield parameters. [0164] At the end of the field trial employing endophyte treatment and control treatment plants, plants (preferably at least 4 plants) are randomly dug out from each row, kept in a plastic bag, and brought back to lab. For each seedling, shoot and root are separated by cutting the seedling 3 cm from the first branch of the root. The heights of the separated shoot of each plant are measured, followed by fresh shoot weight, and fresh root weight. The main root is vertically split into two halves and discoloration of xylem is scored as described above. To extract and count nematode eggs on root, roots are place in a container prefilled with 100 ml 10% sucrose and incubated on a shaker at room temperature overnight. The supernatant is then collected and nematode eggs are counted under a stereomicroscope. [0165] The percentage of survival plants, fresh root weight, and nematode egg count are plotted as bar graph of mean±95% confidence interval from the mean using the ggplot2 package of R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). Plant heights, fresh shoot weight, and disease scores are plotted as jittered dot of mean±nonparametric bootstrap (1000) of 95% confidence interval from the mean using the ggplot2 package of R. Example 14. Field assessment of improved plant health of cotton under biotic stress [0166] This example describes an exemplary method by which improved plant health of endophyte treated plants may be shown in a growth environment comprising the crop pests root knot nematode (Meloidogyne incognita), Reniform nematode (Rotylenchulus reniformis), and, opportunistically, the fungal pathogen Fusarium virguliforme. [0167] Field trials are conducted using chemically treated cotton seeds coated with one or more of the endophytes described herein and formulation control (lacking the one or more heterologously disposed endophytes) and untreated controls (lacking formulation and the one or more heterologously disposed endophyte) as described in Example 4. Plots for in-field assessment harbor populations of root knot nematode and Reniform nematode, respectively, at an approximately 1.0+E04 eggs per gram of fresh root weight. Opportunistically, these plots are infected with natural inoculum of Fusarium virguliforme, the causal agent of Fusarium SDS. Replicate plots, preferably at least 4 replicate plots, are planted per endophyte or control treatment in a randomized complete block design. Each plot consists of a 7.62 m (25 ft.) by 0.76 m (2.5 ft.) row. The following early growth metrics are measured: percent emergence at 14 days post planting, standing count at 28 and 45 days post planting, plant vigor at 14, 28, and 45 days post planting, plant height at 45 days post planting, fresh shoot weight, fresh root weight, disease rating at a 0-3 scale (3 denotes strong disease symptoms) using the split-root scoring system at 45 days post planting, nematode count at 45 days post planting, and yield parameters. [0168] At the end of the field trial employing endophyte treatment and control treatment plants, plants (preferably at least 4 plants) are randomly dug out from each row, kept in a plastic bag, and brought back to lab for metric measurements. For each seedling, shoot and root are separated by cutting the seedling 3 cm from the first branch of the root. The heights of the separated shoot of each plant are measured, followed by fresh shoot weight, and fresh root weight. The main root is vertically split into two halves and discoloration of xylem is scored as described above. To extract and count nematode eggs on root, roots are placed in a container prefilled with 100 ml 10% sucrose and incubated on a shaker at room temperature overnight. The supernatant is then collected and nematode eggs are counted under a stereomicroscope. [0169] The percentage of survival plants, fresh root weight, and nematode egg count are plotted as bar graph of mean±95% confidence interval from the mean using the ggplot2 package of R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). Plant heights, fresh shoot weight, and disease scores are plotted as jittered dot of mean±nonparametric bootstrap (1000) of 95% confidence interval from the mean using the ggplot2 package of R. Example 15. Field assessment of improved plant health of winter wheat under biotic stress [0170] This example describes a method for detection of improved plant health of endophyte treated winter wheat in a growth environment comprising the crop pathogens Rhizoctonia spp., Pythium spp., and Fusarium spp (causal agents of damping-off disease). [0171] Field trials were conducted using winter wheat seeds coated with MIC-84302 and untreated controls (lacking formulation and the heterologously disposed endophyte). Rhizoctonia, Fusarium, and Pythium inoculant were applied per standard practice to each seed packet before planting. Five replicate plots were planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consisted of a 6 ft. by 20 ft. block. Irrigation was applied pre-planting and in early season to maximize disease pressure. [0172] Plots were harvested by machine, and yield was calculated by the on-board computer. Table 9. Yield of endophyte treated winter wheat under biotic stress. Example 16. Field assessment of improved plant health of corn under biotic stress [0173] This example describes a method for detection of improved plant health of endophyte treated corn in a growth environment comprising the crop pathogen Fusarium spp. [0174] Field trials were conducted using corn seeds coated with MIC-67967, a control treated with chemical fungicide (lacking formulation and the heterologously disposed endophyte), and untreated controls (lacking formulation and the heterologously disposed endophyte). Fusarium inoculant was applied per standard practice to each seed packet before planting, targeting moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss. Five replicate plots were planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consisted of a 25 ft. long, 2-4 row block. [0175] Plots were harvested by machine, and yield was calculated by the on-board computer. Table 10. Yield of endophyte treated corn under biotic stress. Example 17. Field assessment of improved plant health of corn under biotic stress [0176] This example describes a method for detection of improved plant health of endophyte treated cotton in a growth environment comprising the crop pathogen Fusarium spp. [0177] Field trials were conducted using cotton seeds coated with MIC-84302, a control treated with chemical fungicide (lacking formulation and the heterologously disposed endophyte), and untreated controls (lacking formulation and the heterologously disposed endophyte). Fusarium inoculant is applied per standard practice to each seed packet before planting, targeting moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss. Five replicate plots are planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consists of a 25 ft. long, 2-4 row block. moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss [0178] Plots were harvested by machine, and yield was calculated by the on-board computer. Table 11. Yield of endophyte treated cotton under biotic stress. Example 18. Field assessment of improved plant health of soybean under biotic stress [0179] This example describes a method for detection of improved plant health of endophyte treated soybean in a growth environment comprising the crop pathogen Pythium sp., Rhizoctonia sp., and Fusarium sp.. [0180] Field trials were conducted using soybean seeds coated with a heterologously disposed endophyte treatment (MIC-54347), a control treated with chemical fungicide (lacking formulation and the heterologously disposed endophyte), and untreated controls (lacking formulation and the heterologously disposed endophyte). Pythium ultimum inoculant was applied in furrow, targeting moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss. Twelve data points were obtained from trials inoculated with the pathogen where stand reduction of at least 5% (or significant loss) occurred in the non-treated control relative to the chemically treated control. At least four replicate plots were planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consisted of approximately a 25 ft. long, 2-4 row block. [0181] Plots are harvested by machine, and yield is calculated by the on-board computer. [0182] Early emergence is the number of emerged plants per acre, measured 0-2 days after the beginning of emergence. Full emergence is the number of emerged plants per acre, measured 10 days after the beginning of emergence. Plant height is the height of five plants per plot, measured 14-21 days after full emergence. Root weight is the weight of roots (cut at the soil line) of five plants per plot, measured 14-21 days after full emergence. Shoot weight is the weight of shoots (cut at soil line) of five plants per plot, measured 17 days after full emergence. [0183] Soybean plants treated with MIC-54347 showed a 13.9% increase in shoot weight over untreated controls, with a 83.5% win-rate. Table 12. Yield of endophyte treated soybean under biotic stress (Pythium) [0184] Field trials were conducted using soybean seeds coated with a heterologously disposed endophyte treatment (MIC-54347), a control treated with chemical fungicide (lacking formulation and the heterologously disposed endophyte), and untreated controls (lacking formulation and the heterologously disposed endophyte). Rhizoctonia solani inoculant was applied per standard practice to each seed packet before planting, targeting moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss. Twelve data points were obtained from trials inoculated with the pathogen where stand reduction of at least 5% (or significant loss) occurred in the non-treated control relative to the chemically treated control (only six data points were obtained for yield data). At least four replicate plots were planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consisted of approximately a 25 ft. long, 2-4 row block. [0185] Plots are harvested by machine, and yield is calculated by the on-board computer. [0186] Early emergence is the number of emerged plants per acre, measured 0-2 days after the beginning of emergence. Full emergence is the number of emerged plants per acre, measured 10 days after the beginning of emergence. Plant height is the height of five plants per plot, measured 14-21 days after full emergence. Root weight is the weight of roots (cut at the soil line) of five plants per plot, measured 14-21 days after full emergence. Shoot weight is the weight of shoots (cut at soil line) of five plants per plot, measured 14-21 days after full emergence. Table 12. Yield of endophyte treated soybean under biotic stress (Rhizoctonia). [0187] Field trials were conducted using soybean seeds coated with a heterologously disposed endophyte treatment (MIC-54347), a control treated with chemical fungicide (lacking formulation and the heterologously disposed endophyte), and untreated controls (lacking formulation and the heterologously disposed endophyte). Fusarium graminearum inoculant was applied in furrow, targeting moderate level of disease infestation; enough to affect plant stand, but not to a level resulting in total loss. Twelve data points were obtained from trials inoculated with the pathogen where stand reduction of at least 5% (or significant loss) occurred in the non-treated control relative to the chemically treated control. At least four replicate plots were planted per endophyte treatment and control treatment in a randomized complete block design. Each plot consisted of approximately a 25 ft. long, 2-4 row block. [0188] Plots are harvested by machine, and yield is calculated by the on-board computer. [0189] Early emergence is the number of emerged plants per acre, measured 0-2 days after the beginning of emergence. Full emergence is the number of emerged plants per acre, measured 10 days after the beginning of emergence. Plant height is the height of five plants per plot, measured 14-21 days after full emergence. Root weight is the weight of roots (cut at the soil line) of five plants per plot, measured 14-21 days after full emergence. Shoot weight is the weight of shoots (cut at soil line) of five plants per plot, measured 14-21 days after full emergence. [0190] Soybean plants treated with MIC-54347 showed a 2.9% (1.77 bu/acre) increase in yield over untreated controls, with a 75% win-rate. Table 12. Yield of endophyte treated soybean under biotic stress (Fusarium). Example 19. Method of preparation of endophytes and heterologous disposition of endophytes on seeds for field trials Preparation of endophytes [0191] Bacteria: An agar plug of each bacterial strain is transferred using a transfer tube to 4 ml of potato dextrose broth (PDB) in a 24 well plate and incubated at room temperature at 675 rpm on a shaker for 3 days. After growth of bacteria in broth, 200 µl is transferred into a spectrophotometer reading plate and bacteria OD is read at 600 nm absorbance. All bacteria strains are then normalized to 0.05 OD utilizing PBS 1x buffer. [0192] Fungi: Preparation of molasses broth and potato dextrose agar: Molasses broth is prepared by dissolving 30 g molasses and 5 g yeast extract per liter deionized water in an autoclavable container and autoclaving (15 psi, 121°C) for 45 min. Potato dextrose agar (PDA) plates are prepared by dissolving 39.0 g PDA powder per liter deionized water in an autoclavable container and autoclaving (15 psi, 121°C) for 45 min. The agar is allowed to cool to 50-60°C, before pouring into sterile petri plates (30 ml per 90 mm plate). Fungal endophyte treatments may be applied as either a dry or liquid formulation. [0193] Liquid biomass: All equipment and consumables are thoroughly sterilized and procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 plug from a cryopreserved stock on a fresh PDA plate, sealing the plate with Parafilm® and incubating at room temperature in the dark for 5-10 days. Then ~5x5 mm plugs are cut from the PDA plates and 10-12 plugs are transferred into flasks containing the sterile molasses broth, covered, secured in a shaker and incubated for at least 10 days with shaking at ~130 rpm. Then the culture is placed in a blender for 5 seconds and 1 ml of the blended culture is centrifuged and the supernatant is discarded. The pellet is resuspended in 0.5 ml 1x Phosphate Buffered Saline (PBS) to generate inoculum. [0194] Dry biomass: All equipment and consumables are thoroughly sterilized and procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 plug from a cryopreserved stock on a fresh PDA plate, sealing the plate with Parafilm® and incubating at room temperature in the dark for 5-10 days. Then ~5x5 mm plugs are cut from the PDA plates and 10-12 plugs are transferred into flasks containing the sterile molasses broth, covered, secured in a shaker and incubated for at least 10 days with shaking at ~130 rpm. In sterile conditions, the liquid culture is carefully decanted using 150 mm sterile filter paper on a sterilized Buchner funnel over a sterile flask. Once all liquid passes through the funnel, the pellet is rinsed with sterile water until the filtrate runs clear. When dry, the pellet is transferred to a drying cabinet and dried until brittle. The pellet is then ground into a fine powder, and sample is used to generate CFU counts. Preparation of formulation for seed treatments [0195] A 2% weight/volume solution of sodium alginate for the seed coatings is prepared by the following method. An Erlenmeyer flask is filled with the appropriate volume of deionized water and warmed to 50 degrees Celsius on a heat plate with agitation using a stir bar. The appropriate mass of sodium alginate powder for the desired final concentration solution is slowly added until dissolved. The solution is autoclaved at 121 degrees Celsius at 15 PSI for 30 minutes to sterilize. [0196] Talc for the powdered seed coatings is prepared by the following method. Talc is aliquoted into bags or 50 ml Falcon tubes and autoclaved in dry cycle (121 degrees Celsius at 15 PSI for 30 minutes) to sterilize. Heterologous disposition of endophytes on seeds [0197] Seeds treated were heterologously disposed to each endophyte according to the following seed treatment protocol. [0198] Liquid formulation: Liquid culture is added to the seeds at a rate of 23 (for fungal endophyte treatments) or 8.4 (for bacterial endophyte treatments) ml per kg of seeds, with equivalent volumes of the prepared sodium alginate. Control treatments are prepared using equivalent volumes of sterile broth. The seeds are then agitated to disperse the solution evenly on the seeds. For fungal endophytes, 15 g per kg of seed of talc powder as prepared above is added and the seeds are agitated to disperse the powder evenly on the seeds. Then 16.6 ml (for fungal endophyte treatments) or 2.4 ml (for bacterial endophyte treatments) per kg of seed of Flo-Rite® 1706 (BASF, Ludwigshafen, Germany) is added and the seeds are agitated to disperse the powder evenly on the seeds. Slightly less Flo-Rite® is used for small grains and canola seeds, slightly more Flo-rite® is used for seeds such as corn, soy, cotton and peanut seeds. The target dose is generally between 10^0 - 10^6 CFU per seed, in some cases at least 10^3 CFU per seed, or at least 10^4 CFU per seed. Treated seeds are allowed to dry overnight in a well-ventilated space before planting. [0199] Dry formulation: The 2% sodium alginate solution prepared above is added to the seeds at a rate of 23 ml per kg of seeds. Equal parts of dry biomass and talc prepared as above are mixed. The solution is applied so that an equivalent of 10 g of powdered dry biomass is applied per kg of seeds. Control treatments are prepared using equivalent volumes of talc. The seeds are then agitated to disperse the solution evenly on the seeds. Then 16.6 ml per kg of seed of Flo-Rite® 1706 (BASF, Ludwigshafen, Germany) is added and the seeds are agitated to disperse the powder evenly on the seeds. Slightly less Flo-Rite® is used for small grains and canola seeds, slightly more Flo-rite® is used for seeds such as corn soy, cotton and peanut seeds. The target dose is generally between 10^0 - 10^6 CFU per seed, in some cases at least 10^3 CFU per seed, or at least 10^4 CFU per seed. Treated seeds are allowed to dry overnight in a well-ventilated space before planting. Example 20. Field assessment of improved plant characteristics Rice [0200] Field trials are conducted, preferably, at multiple locations. In some embodiments, rice seeds are treated with commercial fungicidal and insecticidal treatment. Seeds are heterologously disposed with the endophyte treatments and formulation control (lacking the one or more heterologously disposed endophytes) as described in Example 18, untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted. Seeds are sown in regularly spaced rows in soil at 1.2 million seeds/acre seeding density. At each location at least 3 replicate plots are planted for each endophyte or control treatments in a randomized complete block design. For example, each plot may consist of seven, 15.24 m (40 ft.) rows. [0201] At the end of the field trial employing endophyte treatment and control treatment plants, plots are harvested, for example, by machine with a 5-ft research combine and yield is calculated by the on-board computer. Wheat [0202] Field trials are conducted at multiple locations with multiple plots per location. Wheat seeds (optionally treated with commercial fungicidal and insecticidal treatments) are heterologously disposed with the endophyte treatments as described in Example 18; untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted. Seeds are sown in regularly spaced rows in soil at 1.2 million seeds/acre seeding density. At each location at least 3 replicate plots are planted for each endophyte or control treatments in a randomized complete block design. Each plot consists of seven, 15.24 m (40 ft.) rows. [0203] Plots are harvested by machine, for example with a 5-ft research combine and yield was calculated by the on-board computer. Corn [0204] Field trials are conducted at multiple locations, preferably with multiple plots per location. Plots may be irrigated, non-irrigated (dryland), or maintained with suboptimal irrigation at a rate to target approximately 25% reduction in yield. In some embodiments, corn seeds are treated with commercial fungicidal and insecticidal treatment. Seeds are heterologously disposed with the endophyte treatments as described in Example 18; untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted. Seeds are sown in regularly spaced rows in soil at planting densities typical for each region. At each location at least 3 replicate plots are planted per endophyte or control treatment in a randomized complete block design. For examples, each plot may consist of four 15.24 m (40 ft.) rows, each separated by 76.2 cm (30 in). [0205] At the end of the field trial employing endophyte treatment and control treatment plants, plots are harvested, for example, by machine with a 5-ft research combine and yield is calculated by the on-board computer. Only the middle two rows of the 4 row plots are harvested to prevent border effects. Soy [0206] Field trials were conducted according to the following methodology. Seeds were heterologously disposed with the endophyte treatment (MIC-54347) as described in Example 18, untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted.. At each location at least 3 replicate plots were planted per endophyte or control treatment in a randomized complete block design), a total of 18 data points were collected. Each plot consisted of four 15.24 m (40 ft.) rows, each separated by 76.2 cm (30 in). [0207] At the end of the field trial employing endophyte treatment and control treatment plants, plots were harvested, by machine with a 5-ft research combine and yield is calculated by the on-board computer. Only the middle two rows of the 4 row plots are harvested to prevent border effects. [0208] Treatment with MIC-54347 was associated with a 10.8% decrease in yield where the natural disease pressure in the fields was low, with a win rate of 28%. Canola [0209] Field trials are conducted at multiple locations, preferably in diverse geographic regions. Plots may be irrigated, non-irrigated (dryland) or maintained with suboptimal irrigation at a rate to target approximately 25% reduction in yield. In some embodiments, canola seeds are treated with commercial fungicidal and insecticidal treatment. Seeds are heterologously disposed with the endophyte treatments as described in Example 18, untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted. At each location, at least 3 replicate plots are planted for each endophyte or control treatment in a randomized complete block design. [0210] At the end of the field trial employing endophyte treatment and control treatment plants, plots are harvested, for example, by machine with a 5-ft research combine and yield is calculated by the on-board computer. Peanut [0211] Field trials are conducted at multiple locations, preferably in diverse geographic regions. Optionally, plots are non-irrigated (dryland) or maintained with suboptimal irrigation at a rate to target approximately 25% reduction in yield. In some embodiments, peanut seeds are treated with commercial fungicidal and insecticidal treatment. Seeds are heterologously disposed with the endophyte treatments as described in Example 18, untreated seeds (lacking formulation and the one or more heterologously disposed endophyte) are also planted. [0212] At the end of the field trial employing endophyte treatment and control treatment plants, plots are harvested, for example, by machine with a 5-ft research combine and yield is calculated by the on-board computer. Example 21. Method of determining seed nutritional quality trait component: Fat [0213] Seed samples from harvested plants are obtained as described in Example 20. Analysis of fat is conducted on replicate samples according to the Association of Official Agricultural Chemists Reference Method AOAC 920.39, of the Official Methods of Analysis of AOAC International, 20th Edition (2016), herein incorporated by reference in its entirety. Samples are weighed onto filter paper, dried, and extracted in hot hexane for 4 hrs. using a Soxlhet system. Oil is recovered in pre-weighed glassware, and % fat is measured gravimetrically. Mean percent changes between the treatment (endophyte-treated seed) and control (seed treated with the formulation but no endophyte) are calculated. Example 22. Method of determining seed nutritional quality trait component: Ash [0214] Seed samples from harvested plants are obtained as described in Example 20. Analysis of ash is conducted on replicate samples according to the Association of Official Agricultural Chemists Reference Method AOAC 920.39, of the Official Methods of Analysis of AOAC International, 20th Edition (2016). Samples are weighed into pre-weighed crucibles, and ashed in a furnace at 600ºC for 3hr. Weight loss on ashing is calculated as % ash. Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) with the formulation but no endophyte are calculated. Example 23. Method of determining seed nutritional quality trait component: Fiber [0215] Seed samples from harvested plants are obtained as described in Example 20. Analysis of fiber is conducted on replicate samples according to the Association of Official Agricultural Chemists Reference Method AOAC 920.39, of the Official Methods of Analysis of AOAC International, 20th Edition (2016). Samples are weighed into filter paper, defatted and dried, and hydrolyzed first in acid, then in alkali solution. The recovered portion is dried, weighed, ashed at 600ºC, and weighed again. The loss on ashing is calculated as % Fiber. Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) with the formulation but no endophyte are calculated. Example 24. Method of determining seed nutritional quality trait component: Moisture [0216] Seed samples from harvested plants are obtained as described in Example 20. Analysis of moisture is conducted on replicate samples according to the Association of Official Agricultural Chemists Reference Method AOAC 920.39, of the Official Methods of Analysis of AOAC International, 20th Edition (2016). Samples are weighed into pre-weighed aluminum dishes, and dried at 135ºC for 2hrs. Weight loss on drying is calculated as % Moisture. Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) with the formulation but no endophyte are calculated. Example 25. Method of Determining Seed Nutritional Quality Trait Component: Protein [0217] Seed samples from harvested plants are obtained as described in Example 20. Analysis of protein is conducted on replicate samples according to the Association of Official Agricultural Chemists Reference Method AOAC 920.39, of the Official Methods of Analysis of AOAC International, 20th Edition (2016). Samples are combusted and nitrogen gas is measured using a combustion nitrogen analyzer (Dumas). Nitrogen is multiplied by 6.25 to calculate % protein. Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) with the formulation but no endophyte) are calculated. Example 26. Method of determining seed nutritional quality trait component: Carbohydrate [0218] Seed samples from harvested plants are obtained as described in Example 20. Analysis of carbohydrate is determined for replicate samples as a calculation according to the following formula: Total Carbohydrate = 100% - % (Protein + Ash + Fat + Moisture + Fiber), where % Protein is determined according to the method of Example 25, % Ash is determined according to the method of Example 22, % Fat is determined according to the method of Example 21, % Moisture is determined according to the method of Example 24, and % Fiber is determined according to the method of Example 23. Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) are calculated. Example 27. Method of determining seed nutritional quality trait component: Calories [0219] Seed samples from harvested plants are obtained as described in Example 20. Analysis of Calories is determined for replicate samples as a calculation according to the following formula: Total Calories = (Calories from protein) + (Calories from carbohydrate) + Calories from fat), where Calories from protein are calculated as 4 Calories per gram of protein (as determined according to the method of Example 25), Calories from carbohydrate are calculated as 4 Calories per gram of carbohydrate (as determined according to the method of Example 26), and Calories from fat are calculated as 9 Calories per gram of fat (as determined according to the method of Example 21). Mean percent changes between the treatment (one or more heterologously disposed endophytes) and control (lacking the one or more heterologously disposed endophytes) are calculated. Example 28. Additional methods for creating synthetic compositions Osmopriming and Hydropriming [0220] One or more endophytes are inoculated onto seeds during the osmopriming (soaking in polyethylene glycol solution to create a range of osmotic potentials) and/or hydropriming (soaking in de-chlorinated water) process. Osmoprimed seeds are soaked in a polyethylene glycol solution containing one or more endophytes for one to eight days and then air dried for one to two days. Hydroprimed seeds are soaked in water for one to eight days containing one or more endophytes and maintained under constant aeration to maintain a suitable dissolved oxygen content of the suspension until removal and air drying for one to two days. Talc and or flowability polymer are added during the drying process. Foliar application [0221] One or more endophytes are inoculated onto aboveground plant tissue (leaves and stems) as a liquid suspension in dechlorinated water containing adjuvants, sticker-spreaders and UV protectants. The suspension is sprayed onto crops with a boom or other appropriate sprayer. Soil inoculation [0222] One or more endophytes are inoculated onto soils in the form of a liquid suspension, either; pre-planting as a soil drench, during planting as an in-furrow application, or during crop growth as a side-dress. One or more endophytes are mixed directly into a fertigation system via drip tape, center pivot or other appropriate irrigation system. Hydroponic and Aeroponic inoculation [0223] One or more endophytes are inoculated into a hydroponic or aeroponic system either as a powder or liquid suspension applied directly to the rockwool substrate or applied to the circulating or sprayed nutrient solution. Vector-mediated inoculation [0224] One or more endophytes are introduced in power form in a mixture containing talc or other bulking agent to the entrance of a beehive (in the case of bee-mediation) or near the nest of another pollinator (in the case of other insects or birds. The pollinators pick up the powder when exiting the hive and deposit the inoculum directly to the crop’s flowers during the pollination process. Root Wash [0225] The method includes contacting the exterior surface of a plant’s roots with a liquid inoculant formulation containing one or more endophytes. The plant’s roots are briefly passed through standing liquid microbial formulation or liquid formulation is liberally sprayed over the roots, resulting in both physical removal of soil and microbial debris from the plant roots, as well as inoculation with microbes in the formulation. Seedling Soak [0226] The method includes contacting the exterior surfaces of a seedling with a liquid inoculant formulation containing one or more endophytes. The entire seedling is immersed in standing liquid microbial formulation for at least 30 seconds, resulting in both physical removal of soil and microbial debris from the plant roots, as well as inoculation of all plant surfaces with microbes in the formulation. Alternatively, the seedling can be germinated from seed in or transplanted into media soaked with the microbe(s) of interest and then allowed to grow in the media, resulting in soaking of the plantlet in microbial formulation for much greater time, for example: hours, days or weeks. Endophytic microbes likely need time to colonize and enter the plant, as they explore the plant surface for cracks or wounds to enter, so the longer the soak, the more likely the microbes will successfully be installed in the plant. Wound Inoculation [0227] The method includes contacting the wounded surface of a plant with a liquid or solid inoculant formulation containing one or more endophytes. Plant surfaces are designed to block entry of microbes into the endosphere, since pathogens attempt to infect plants in this way. One way to introduce beneficial endophytic microbes into plant endospheres is to provide a passage to the plant interior by wounding. This wound can take a number of forms, including pruned roots, pruned branches, puncture wounds in the stem breaching the bark and cortex, puncture wounds in the tap root, puncture wounds in leaves, puncture wounds seed allowing entry past the seed coat. Wounds can be made using tools for physical penetration of plant tissue such as needles. Microwounds may also be introduced by sonication. Into the wound can then be contacted the microbial inoculant as liquid, as powder, inside gelatin capsules, in a pressurized capsule injection system, or in a pressurized reservoir and tubing injection system, allowing entry and colonization by microbes into the endosphere. Alternatively, the entire wounded plant can be soaked or washed in the microbial inoculant for at least 30 seconds, giving more microbes a chance to enter the wound, as well as inoculating other plant surfaces with microbes in the formulation – for example pruning seedling roots and soaking them in inoculant before transplanting is a very effective way to introduce endophytes into the plant. Injection [0228] The method includes injecting microbes into a plant in order to successfully install them in the endosphere. Plant surfaces are designed to block entry of microbes into the endosphere, since pathogens attempt to infect plants in this way. In order to introduce beneficial endophytic microbes to endospheres, we need a way to access the interior of the plant which we can do by puncturing the plant surface with a needle and injecting microbes into the inside of the plant. Different parts of the plant can be inoculated this way including the main stem or trunk, branches, tap roots, seminal roots, buttress roots, and even leaves. The injection can be made with a hypodermic needle, a drilled hole injector, or a specialized injection system, and through the puncture wound can then be contacted the microbial inoculant as liquid, as powder, inside gelatin capsules, in a pressurized capsule injection system, or in a pressurized reservoir and tubing injection system, allowing entry and colonization by microbes into the endosphere. Example 29. Identification of sequence variants across core genes [0229] Phylogenomic analysis of whole genome sequences of endophytes can be used to identify distinguishing sequence variants. Sets of genes suitable for phylogenomic analysis as well as methods for identifying the same are well known in the art, for example Floutas et al. (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science, 336(6089):1715-9. doi: 10.1126/science.1221748 and James TY, Pelin A, Bonen L, Ahrendt S, Sain D, Corradi N, Stajich JE. Shared signatures of parasitism and phylogenomics unite Cryptomycota and microsporidia. Curr Biol.2013;23(16):1548-53. doi: 10.1016/j.cub.2013.06.057. Orthologous genes to the reference set are identified in protein data bases derived from the genome of each species. Orthologous genes can be identified in the genomes using methods well known including reciprocal best hits (Ward N, Moreno- Hagelsieb G. Quickly Finding Orthologs as Reciprocal Best Hits with BLAT, LAST, and UBLAST: How Much Do We Miss? de Crécy-Lagard V, ed. PLoS ONE.2014;9(7):e101850. doi:10.1371/journal.pone.0101850) and Hidden Markov Models (HMMs). The best hits are extracted and a multiple sequence alignment generated for each set of orthologous genes. The alignments are used to build phylogenetic trees using methods well known in the art including Bayesian inference and maximum likelihood methods, for example using software tools MrBayes (Huelsenbeck, J.P. & Ronquist (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17(8):754-755) and RAxML (Stamatakis, A. (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30 (9): 1312-1313. doi: 10.1093/bioinformatics/btu033). Sequence variants which distinguish between closely related species are identified. Example 30. Identification of unique genes in an endophyte of interest [0230] Whole genome analysis of endophytes can be used to identify genes whose presence, absence or over or under representation (“differential abundance”) are associated with desirable phenotypes. To identify genes with differential abundance in the genome of an endophyte of interest, protein sequences predicted from the genomes of the endophyte and closely related species are compared in an all-vs-all pairwise comparison (for example, using BLAST) followed by clustering of the protein sequences based on alignment scores (for example, using MCL: Enright A.J., Van Dongen S., Ouzounis C.A. An efficient algorithm for large-scale detection of protein families. Nucleic Acids Research 30(7):1575-1584 (2002)). Additional software tools useful for this analysis are well known in the art and include OMA, OrthoMCL and TribeMCL (Roth AC, Gonnet GH, Dessimoz C. Algorithm of OMA for large-scale orthology inference. BMC Bioinformatics.2008;9:518. doi: 10.1186/1471-2105- 9-518, Enright AJ, Kunin V, Ouzounis CA. Protein families and TRIBES in genome sequence space. Nucleic Acids Res.2003;31(15):4632-8.; Chen F, Mackey AJ, Vermunt JK, Roos DS. Assessing performance of orthology detection strategies applied to eukaryotic genomes. PLoS One.2007;2(4):e383.). The protein clusters are queried to identify clusters with differential abundance of proteins derived from endophytes having desirable phenotypes. Proteins of these clusters define the unique properties of these endophytes, and the abundance of genes encoding these proteins may be used to identify endophytes of the present invention. Example 31. In vitro assessment of production of antibiotic metabolites [0231] This example describes an exemplary method by which microbes may be shown to produce metabolites that inhibit the growth of hyphal phytopathogens in vitro. Such phytopathogens can be members of the “true” fungi, phylum Eumycota, or from other taxonomic groups with a similar growth habit such as members of the phylum Oomycota. Hyphal growth can be described as organism growth along thread-like structures composed of connected cells. Such growth is found commonly among fungi and oomycetes, and even some genera of bacteria. In this assay, the hyphal growth should be in a roughly uniform, radial manner. This assay is comprised of a Petri plate containing an agar-based media and a hyphal phytopathogen grown concomitantly with either a live test microbe or in the presence of the spent media from a previously grown test microbe. Testing with live endophyte cultures [0232] Preparation of Hyphal Phytopathogen Petri plates containing a media suitable for the growth of the target hyphal pathogens (Fusarium graminearum, Rhizoctonia solani, Pythium sp.) were inoculated with the target hyphal pathogen. After inoculation on the media- containing Petri plate, the culture was allowed to grow until reaching the edge of the Petri plate. [0233] Preparation of the test sample Microbial samples for testing endophytes MIC-84302, MIC-18905, and MIC-67967 were produced by liquid culture. [0234] Assay Set-Up Petri dishes, also referred to as test plates, containing solid agar test media (see Use of Multiple Growth Media for a description of media used) were prepared. A sterile instrument was used to remove a test pathogen plug from the hyphal pathogen plate culture and placed centrally on the test plate. Next a test sample was applied to the test plate at a distance such that the test sample and test plate came into physical contact after more than one day of growth. A drop of overnight liquid culture of the endophyte to be tested was applied to each test plate. A drop of Metconazole, a chemical fungicide capable of impeding the growth of Fusarium and Rhizoctonia was applied to each test plate containing those pathogens as a control. A drop of Mefenoxam, a chemical fungicide capable of impeding the growth of Pythium was applied to each test plate containing Pythium. For an example of the relative position of the test sample, pathogen sample, and chemical control refer to Fig.1C, . [0235] Use of Multiple Growth Media Various environmental conditions can result in differential production of metabolites and pathogens grown under various environmental conditions show differential sensitivity to those metabolites, therefore the assay was performed on multiple media types: half strength Potato Dextrose Agar (0.5x PDA), yeast extract peptone dextrose agar (YEPD), tryptic soy agar (TSA), and Reasoner’s 2A agar (R2A). Medias were chosen to vary important growth inputs such as carbon source, presence and concentration of various salts, and presence of extracts from different plant species or organs. [0236] Assessment After setting up, hyphal pathogens were allowed to grow for sufficient time such that the hyphal front meets or just passes the test sample. In cases where anti- pathogen metabolites are produced and secreted, a restriction of growth of the hyphal front around the test sample is commonly observed. Often this will also result in an area of clearing around the test sample. In these cases, the morphology of the hyphal pathogen near the test sample will often also be dissimilar from areas away from the test sample. Alternatively, when anti-pathogen metabolites are not produced and secreted, the hyphal pathogen will grow over the test sample with little to no visible effect on growth. [0237] Exemplary images of test plates treated with endophytes, chemical fungicides and pathogens are shown in Fig.1A-C, Fig.2A-B, Fig.3A-C, Fig.4A-B, Fig.5A-C, and Fig.6A- B. [0238] MIC-18905 and MIC-84302 showed significant pathogen-free zones around the test samples of these endophytes on TSA test plates inoculated with Fusarium graminearum (see for example Fig.1A, Fig.1B, and Fig.1C). MIC-67967 showed pathogen-free zones around the test samples of this endophyte on R2A test plates inoculated with Fusarium graminearum (see for example Fig.2A and Fig.2B). [0239] MIC-18905 and MIC-84302 showed significant pathogen-free zones around the test samples of these endophytes on 0.5xPDA test plates inoculated with Rhizoctonia solani (see for example Fig.3A, Fig.3B, and Fig.3C). MIC-67967 showed pathogen-free zones around the test samples of this endophyte on 0.5xPDA test plates inoculated with Rhizoctonia solani (see for example Fig.4A and Fig.4B). [0240] MIC-18905 and MIC-84302 showed significant pathogen-free zones around the test samples of these endophytes on 0.5xPDA test plates inoculated with Pythium (see for example Fig.5A, Fig.5B, and Fig.5C). MIC-67967 showed significant pathogen-free zones around the test samples of this endophyte on YEPD test plates inoculated with Pythium (see for example Fig.6A and Fig.6B). Testing with filtered or dead endophyte cultures [0241] Pathogen samples are prepared as described above. A microbial sample for testing, also referred to as a test sample, can be produced in multiple ways. A liquid culture of hyphal or colony forming microbe is grown in liquid culture, and viable material is removed by various methods including, but not limited to, filtration. Alternately, or in addition to filtration a test sample may be autoclaved and a non-viable test sample may be used. This later method of testing a non-viable test sample is used when the test microbe displays a much faster rate of radial growth than the hyphal pathogen being tested, to identify production of antimicrobial metabolites, for example not as a part an active biological process such a mycophagy. [0242] Assay Set-Up A Petri dish containing a solid agar test media is obtained. This will be referred to as the test plate. A sterile instrument is used to remove a test pathogen plug from the hyphal pathogen plate culture and placed on the test plate. For assaying a non-viable test sample, an agar plug is removed from the test plate using a sterile instrument to create a well to hold the test sample. The well is then filled with the non-viable test sample, and the sample is absorbed into the agar media. A chemical compound capable of impeding the growth of the pathogen is included as a control. For an example of the relative position of the test sample, pathogen sample, and chemical control refer to Fig.1. [0243] Use of Multiple Growth Media Test microbe growth under various environmental conditions are expected to result in differential production of metabolites. Similarly, pathogens grown under various environmental conditions are expected to show differential sensitivity to those metabolites. For this reason, this assay is performed on multiple media types. Medias are chosen to vary important growth inputs such as carbon source, presence and concentration of various salts, and presence of extracts from different plant species or organs. [0244] Assessment After setting up, hyphal pathogens are allowed to grow for sufficient time such that the hyphal front meets or just passes the test sample. In cases where anti-pathogen metabolites are produced and secreted, a restriction of growth of the hyphal front around the test sample is commonly observed. Often this will also result in an area of clearing around the test sample. In these cases, the morphology of the hyphal pathogen near the test sample will often also be dissimilar from areas away from the test sample. Alternatively, when anti- pathogen metabolites are not produced and secreted, the hyphal pathogen will grow over the test sample with little to no visible effect on growth. [0245] Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other embodiments, advantages, and modifications are within the scope of the following claims.