GanoEFl FIELD OF INVENTION

[0001] The present invention relates to methods for controlling plant diseases and more particularly to the identification and isolation of a fungal species with biological activity against Ganoderma diseases, and compositions comprising said fungal species.

BACKGROUND OF INVENTION [0002] It is generally agreed that scientific aspects play a major role in contributing to improvements of various crop traits and cultivation to which such researches have become vital for the experts of the agricultural industry, and much has been written about them.

[0003] In most cases, the science of manipulation in plants is focused on improvements of quality or high-value products, diversification, and in particular for assisting in improving or achieving increased total yield of crops or plants, in addition to obtaining desired vegetative characters and attributes. Another imperative aspect in view of agricultural economic development is disease management.

[0004] In recent years, one of the various plant diseases widely known to be a major threat particularly for oil palm cultivation is the Basal Stem Rot (BSR) . There has been a general consensus within the oil palm industry that BSR is the primary route of infection. Findings have shown that BSR is generally caused by a species.. of Ganoderma, a wood decaying fungus. Ganoderma produces enzymes that degradesthe... oil. palm . tissue, .and.. gradually affects the infected oil palm xylem thus resulting to severe problems to the distribution of water and other nutrients to the top of palm tree (Paterson et al, 2008, Current Enzyme Inhibition) . Consequently, the palm will lose its ability to produce fruits and will eventually collapse.- Infections have been reported to be rapidly increasing in numerous estates in many countries and some reports have shown that younger palms of around 5 years old can also be affected.

[0005] As a preliminary effort in developing a solution to resolve this growing concern, extensive studies have enabled researchers to successfully identify four species of Ganoderma associated with BSR disease of oil palm; these are G. boninense, G. zonatum, G. miniatocinctum and G. tornatum . Among all four, G. boninense has been identified as the most virulence and most annihilating in Malaysia and several South East Asian countries.

[0006] Microbial based agents have shown significant effects against plant diseases; whereby there are several prominent microbials identified and characterised to have shown antagonistic properties against G. boninense, such as Trichoderma , Aspergillus, and Penicillium . [0007] Endophytes, more often bacteria or fungi live within .the. intracellular spaces of plants for at least all of their life cycles without causing any harmful diseases to their host plants. These fungi live off apoplastic nutrients which are naturally occurring in plants. Some fungi can be isolated from host plant families while others are restricted to host species within a particular plant family. Recent findings (Marciano et al, 2005) have shown that a type of endophytic fungus, Gliocladium catenulatum exhibited antagonistic properties against Witches Broom disease caused by Crinipelis perniociosa in cacao seedlings. Although the use of endophytes has been . disclosed for such specific disease, there is still dire need to identify and thus characterise more plant based fungus for the purpose of disease management associated to various plants.

[0008] Because oil palm is one of the most important commercial crops in Malaysia, it has been identified as the most likely candidate ..-for the development of a large scale production and renewal plant (Ravigadevi et al, Genetic engineering in oil palm. In Advances in Oil Palm Research. (eds) . Yusof , Jalani, and Chan Malaysia Palm Oil Board. 2:284-331, 2000) for palm oil-derived chemicals. As discussed earlier, unrestrained spread of diseases infection in oil palm cultivation will gradually cause a significant impact on the economic growth of the industry.

[000.9] Eallojw.ing .the above, . there is. a .need to identify a

-method to address the glaring issues with respect to oil palm, diseases, and in particular Basal. Stem Rot (BSR) dis ase,.. _^

SUMMARY OF THE INVENTION

[0010] In one aspect, the invention provides a method for controlling Ganoderma associated diseases, said method includes providing a composition comprising nutrient supplemented granules of viable fungal propagules of an endophytic fungus.

[0011] In another aspect, the invention provides an isolated culture of a fungus identified as Hendersonia GanoEFl used for the preparation of the composition. [0012] Another aspect of the invention provides a method, for treating or controlling Ganoderma disease infection in oil palm.

[0013] In another aspect, the invention provides a method for treating or controlling Basal Stem Rot (BSR) disease using a composition comprising viable fungal propagules of an endophytic fungus, said fungus from the Hendersonia genus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] h cure 1......depicts rie, .pe centage of disease incidence (DI%) of seedlings due to G. boninense infection at six months of treatment with the composition of the present invention;

[0015] Figure 2 depicts the percentage of severity of foliar symptoms (%SFS) of seedlings due to G. boninense infection at six months of treatment with the composition of the present invention; and

[0016] Figure 3 depicts the percentage of dead seedlings (%DS) due to the infection of G. boninense infection at six months of treatment with the composition of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Further understanding of the object, construction, characteristics and functions of the invention, a detailed description with reference to the embodiments is given in the .following.

[0018].. Embodiments.- of the present. invention are. predicated in part on the identification and isolation of an endophytic fungus which.. is useful .in ..a method for.controlling Ganoderma diseases in oil palm plant.

[0019] The composition according to a preferred embodiment of the present invention may be prepared based on a pure culture of an endophytic fungus named as Hendersonia GanoEFl. The composition may be in the form of, but not limiting to granules, or a bio-fertilizer.

[0020] In accordance to the preferred embodiment of the present invention, there is disclosed a method for producing the formulated forms of the fungus as identified above. [0021] The present disclosure of the present inventionprovides an endophytic fungus that has been identified to exhibit substantial effects in controlling the BSR infection in oil palm. It is observed that the composition comprising said fungus showed significant reduction of Disease Incidence (%) in germinated seedlings of oil palm.

[0022] The present disclosure further provides the isolation of a fungus identified as. Hendersonia GanoEFl, whereby said fungus has unusual effects against BSR, and having unique inoculants feature in controlling Ganoderma disease of- oil pal .-

[0023] It,, is .observed "hat. the ...profil of. Hendersonia GanoEFl fungus has been identified to have high similarity with Neoscytalidium dimidiatum (DQ 377924.1). Although the Neoscytalidium genus has been reported to be recognized as one of the plant pathogen fungi, however there has yet to be any prior isolates of a similar genus or reports that are associated to this category of fungi as a cure to oil palm diseases, or used in controlling oil palm diseases. While Hendersonia is known to be associated with the Coelomycetes genus, and so far has yet to be reported as part of a cure or composition for controlling or inhibiting infections of diseases in oil palm. [0024] Characterization of Hendersonia GanoEFl A standard sequence analysis was performed to identify the DNA profile of the fungus used in accordance with the present invention. As briefly discussed above, it is observed that the profile of the fungus has high similarity to that of Neoscytalidium dimidiatum (DQ377924.1 - NCBI) . In this analysis, twenty primers (OPl to OP20) were screened in order to identify the DNA markers for the identification of the fungus . Three markers have shown intact bands in agarose gel image thus indicates the profile of the fungus isolated .in. accordance with the present.. invention .

In accordance . .with the present - invention, .. using LALIGN .so-f; Lwar.e .ht-tp.: / /www., eh-;.embne.t- o.rq/soft.ware/LALIGN form, html ) the subject Hendersonia GanoEFl has been found 44% similar to Pleospora sp . strain LM-2009a Internal Transcribed Spacer 1, Partial sequence, 5.8S Ribosomal RNA gene, Internal Transcribed Spacer 2, Partial sequence anamorph Hendersonia osteospermi ( FJ 81946.1 ) . The similarity search was done by using BLOSU 62 matrix scoring and local alignment (matrix file: /usr/molbio/share/fasta3/blosum62. mat (11/-4), gap- open/ext: -14/-4 E(limit) 0.05). About 577 nucleotides were overlapping (1-541:435-1006), score is 1008 E (10000):- 1 The sequence obtained is as follows: , ^^^M ^^&^^^^^ss ^^^^^^^^^^^ ^ m

ITGTTGGT-TTCTTTTCCGCCGCTTATTGA

EXAMPLES-

- [0025] The method of preparation and. use of the- present invention is further illustrated ^; by the following experimental examples. It should be understood that these experimental examples, while indicating preferred embodiments of the invention, are given by way better elucidation only. A person skilled in the art can ascertain the essential characteristics and embodiments of this invention, therefore various changes may be provided to adapt to various usages and conditions. [0026] EXAMPLE 1: Preparation of Nutrient Supplemented

Granules based on Hendersonia GanoEFl .· . / ^"

[ 0027 ] Fungal spores were propagated in 1 liter (L) conical flasks which contained 250 mililiter (mL) of liquid media. In this experiment, the preferred media comprised of palm oil mill effluent (POME) and 9% by weight per volume (w/v) of jaggery. The flasks were autoclaved at a temperature of 1.20°C. for .20 minutes and inoculated by adding spore suspension which contained about 10 ⁸ spores per mL (spores/rr.1) , . whereby said .spares were prepared based on 12 days old cultures. The flasks were then placed on a orbital..shaker and. ran ..at 150 revolutions . per. minute, (rpm).. for four -days _: . _. ..a-t ;.hc. r.e~pe a~ure.. of ..3.0 °C. The resultant ,. viable propagules were used to. prepare the granules which will be described herein below.

[0028 ] EXAMPLE 2: Preparation of Formulated form of Fungus -Granules

[ 0029] The formulated form of the fungus may be prepared with a method which will be described herein, whereby it is apparent to a person skilled in the art that the sequence of the steps involved may vary to achieve the intended result however not exceeding the scope of claims as provided. [0030] The first step was preparing alginate solution which contains about 1% (w/v) alginic acid by dissolving sodium alginate into absolute alcohol. Then the alginate solution was mixed with distilled water until dissolved. Propagules grown in 250mL flask were homogenized so as to break up the mycelia into small fragments. The homogenate mycelia and spores were mixed with the alginate solution prepared earlier thus accumulated to a total of 2L solution. Next, . about 20% (w/v) of kaolin, and. 10% (w/v) of dhal were added slowly into the homogenate obtained earlier. It should be., noted-that ..kaolin... and. dhal ...were . auto.cla.ved. prior . to being added so as to- prevent contamination. Granulation process of the present invention involved the use of a peristaltic pumpattached with micropipette tips. The mixture was reduced into droplets by means of the micropipette, whereby said droplets were introduced into a container containing coagulant agent and round shaped granules were formed during this stage. Within 2 to 4 minutes, the granules were rinsed with sterilized distilled water and later on dried in laminar flow for 24 hours.

[0031] Dhal was added to supply nutrient substrates, carbon and nitrogen sources to the fungus for long lasting sporulation to the granules especially when it is applied on plants. Kaolin on the other hand was added to as to provide density to the granules of the present invention and thus acts as an absorber material to absorb moisture from moist substrates. Moisture is a growth factor for mycelia and .for producing spores. [0032 ] The preferred coagulant used for the preparation of formulated composition in accordance with the preferred embodiment of the present invention is Calcium Chloride (CaCl ₂ ) , whereby the concentration is about 0.5% (w/v) . [0033] The dried granules prepared in accordance with the method of the present. _ invention ..can be stored under dry conditions at room temperature .

[0034 ] According. ^* .,o the .preferred, embodiment... of the present invention, - the granules are preferably ^" applied direct-ly on roots of the oil palm to promote colonization into oil palm roots. During application, whereby the preferred method is to allow contact with seedlings and planting holes, granules will absorb sufficient moisture to initiate the growing process. The granules will start producing the spores after 4 to 5 days after the fungal growth .

[0035] The composition as described above was tested on seedlings of oil palm, whereby said seedlings were treated with the granules of the present invention and inoculated with G. boninense . The composition was applied periodically based on the growth age of the; oil palm.

[0036] EXAMPLE 3: Analysis on the Effects of the Composition on Germinated Seedlings of Oil Palm

[0037] 15 grams Hendersonia GanoEFl granules formation as prepared in the previous section was applied into the root of the germinated seedlings., while transferring to the small polyethylene bag sized 6 x 9 cm to promote the colonization into...the .-oil. palm roots.. .At four., month old, the. seedlings were- transferred to the big -polyethylene bag sized 15 x 18 cm. and .50. grams of Hendersonia GanoEFl. granules was .applied, ^" .'he.. Lrea::ed .and... ntreated seedlings .. were r er. . inoculated with G. boninense block using Rubber Wood Block (RWB) sitting technique-. At subsequent months, 30 grams of Hendersonia GanoEFl granules was applied to the treated seedlings.. All the seedlings were maintained in the nursery, supplemented with normal fertilizer and watered daily. Seedlings untreated with the composition of the present invention and inoculated with G. boninense were prepared as Control Samples.

[0038] Six months after treatment, seedlings treated with the composition comprising Hendersonia GanoEFl shows a lower percentage of Disease Incidence (%DI) as plotted in FIG 1. Disease development was also monitored based on the .percentage of Severity of Foliar Symptoms (%SFS) .

[0039] It is further observed that there were significant differences between seedlings treated with the composition of the present invention and untreated seedlings, at 6 months, as shown in FI 2. In the case of deadline seedlings percentage, untreated seedlings showed the highest percentage of dead seedlings and the amount of dead seedling in treated seedlings was significantly reduced. This _. is evidently shown in FIG ^' 3.

[0040] As seen in FIG 1, samples treated with the ■ composition .. comprising ...Hendersonia... Ganci'lFl . of the present invention exhibited at least 60% - of disease incidence -reduction as compared to untreated samples. It is observed that seedling treated with Hendersonia GanoEFl granules showed a significant (p<0.05) lower percentage of disease incidence (DI) at average 39.2% compared to untreated seedlings at 95%. In FIG 2, the percentage of severity of foliar symptoms (SFS) of treated samples was reduced by at least 50% in percentage when compared to untreated samples. Accordingly, the average percentage SFS of seedlings treated with Hendersonia GanoEFl granules (54.5%) was significantly lower (p<0.05) than that of the untreated seedlings. It is observed that the average percentage. SFS of seedlings treated with Hendersonia GanoEFl granules (54.5%) was significantly (p<0.05) lower compared' to the untreated seedlings (90.5%) . The percentage of dead seedlings due to G boninense infection was observed to be considerably reduced for treated samples, whereby at least 70% reduction was recorded. For this, it is observed that the average percentage of dead seedlings in control showed the highest percentage of dead seedlings at 80.8% and significantly (p<0.05). as compared to the., seedlings treated with Hendersonia GanoEFl granules at 34.2%.

[0041] Further, the samples treated with the composition prepared, in . accordance with the. . present invention gave lowest -Ar- ₌ ea-,Und.e ----Diseas-e..---Erogj:ess--^Curve- _^ -tAUD-PC-^-af,. -.86.6 and 89.0 in two trials, compared to untreated seedlings with AUDPC of 193.3 and 190.0 for both trials respectively. TABLE 1 below provides a summary on the AUDPC comparison between the treated samples and untreated samples as previously discussed :

[0042] TABLE 1: Effect of Hendersonia GanoEFl Granules on

Basal Stem Rot (BSR) Disease Development in Oil Palm

Seedlings at Six Months After Treatment Trial 1 Trial 2

.Treatments AUDPC* DR ^# * AUDPC* DR ^# *

(%) (%)

Seedlings untreated with

Hendersonla. GanoEFl granules 193.34 — 190.0 — and inoculated with

G. boninense (control)

Seedlings treated with

Hendersonxa GanoEFl granule

and inoculated with G. 86.67 55.17 89.0 53.15 boninense (Tl)

Note: Area Under Disease Progress Curve (AUDPC) .

Disease Reduction (DR).

Average disease reduction (DR) = 54.16% [0043] It is concluded that the composition comprising Hendersonla GanoEFl successfully controlled the Ganoderma infection on samples of seedlings treated with said composition, as evident in the graphs of FIG 1 to FIG 3. _. Understandably, the composition of the present invention may be used to inhibit or prevent further infection of Ganoderma in oil palm.

[0044] The composition of the present invention may be effective for the treatment of Ganoderma infected oil palms, whereby the composition aids to control the infection of Ganoderma in oil palms. It should be noted that the Ganoderma species may be any one from the group of G. boninense , G. zonatum, G. miniatocintum and G. tornatum .

[0045] Although the present invention has been described with reference to the preferred embodiments and examples thereof, it is apparent to. those skilled in the art that ava iet _∞^ o,f . modifications .and. changes may .be.- ...made.-_..without departing from the scope of the present invention which is intended to be defined by the appended claims.