WO2014133378A1

WO2014133378A1 - Compositions for controlling ganoderma disease in plants and method thereof by using soil actinomycetes

Info

Publication number: WO2014133378A1
Application number: PCT/MY2014/000022
Authority: WO
Inventors: Abu Seman IDRIS; Muzaimah Syed Aripin SHARIFFAH; Ahmad Zairun MADIHAH; Kushairi Din AHMAD
Original assignee: Malaysian Palm Oil Board
Priority date: 2013-02-27
Filing date: 2014-02-26
Publication date: 2014-09-04
Also published as: MY180774A

Abstract

The present invention discloses a composition, method of preparation of said composition and method of biologically controlling fungal infection in plants, particularly the basal stem rot disease primarily caused by Ganoderma species. Said composition comprises of actinomycetes bacteria from Streptomyces species, and one type of microbial carrier.

Description

COMPOSITIONS FOR CONTROLLING GA ODERMA DISEASE IN PLANTS AND METHOD THEREOF BY USING SOIL ACTINOMYCETES

FIELD OF INVENTION

The present invention relates to a composition and method for controlling fungal infection in plants. More particularly, relates to the use of soil actinomycetes in controlling Ganoderma infection in plants.

BACKGROUND OF INVENTION

Ganoderma infection, particularly basal stem rot (BSR) disease is caused by Ganoderma species. Ganoderma fungi are basidiomycetes where three pathogenic species are reported to be responsible for BSR disease. The three species are G. boninense, G. zonatum, and G. miniatocinctum , in which Ganoderma boninense is identified as the most pathogenic and causes severe infection in oil palm among all known species. It is reported that it has higher risk of infection in replanted areas from oil palm and coconut. BSR is a major and serious disease prevalent in oil palm that can lead to low productivity and death of plants thus resulting in loss to the producer causing losses up to several hundred thousand dollars per hectares. Currently, there are several techniques proposed for controlling Ganoderma infection in oil palm. Chemical treatment with synthetic fungicide based on triazole group, such as bromoconazole and hexaconazole has been widely employed in oil palm. These two fungicides however are only able to prolong the life span of infected palm but have failed to inhibit Ganoderma fungus from growing and in preventing the infection from spreading. In vitro studies by Idris et al. (2002) claimed that numerous fungicides such as benomyl, triadimefon, cycloheximide and drazoxolone strongly inhibit the growth of Ganoderma; however the efficiency of these fungicides on the field is still inconclusive. These chemical treatments are impractical for large scale application, primarily due to its high cost of implementation. Furthermore, they are not environmental-friendly and might have adverse effects on health .

An example relates to composition for use in treating, inhibiting or preventing the development of BSR disease caused by G. boninense is disclosed in prior art document O2010115162. The composition comprises volatile organic compounds selected from a group consisting of isobutyric acid, methyl isobutyrate, isobutyric anhydride, ethyl isobutyrate, propyl isobutyrate, isobutyric acid-allyl ester, naphthalene, caryophyllene to inhibit the growth of G. boninense . This fungicide composition is effective in protecting oil palms from Ganoderma infection. However, undesirable health and environmental effects may accompany the use of said fungicide.

Azizah (2003) reported that, seedlings inoculated with microbes were found to be protected from Ganoderma. Plant house trial by using isolate T. harzianum FA 1132 done by Abdullah et al. (1999) has shown good antagonist properties against G. boninense and is effective in controlling disease development (Izzati and Abdullah 2008) . Biological control using Trichoderma spp. showed a high efficacy in controlling the growth and infection by G. boninense in plant house trials and under field conditions (Abdullah et al. 1999; Ilias 2000; Sariah et al. 2005; Susanto et al. 2005). Apart from the Trichoderma spp., studies using other microbes such as Mychorriza , Gliocladium viridae and Bacillus spp. are thoroughly investigated as well (Arifin et al. 1996; Azizah, 2003). According to Tan et al. 2002, Streptomyces genus in actinomycetes group showed a high inhibition percentage towards G. boninense in vitro through antibiosis and the lysis of hyphae resulting in abnormal hyphae growth. Actinomycetes are common filamentous gram positive bacteria which lived in soil and rhizosphere. They were reported to be a major source of useful secondary metabolites. These biological active substances include antibiotics such as alnumycin, polyene antibiotics, geldanamycin (Kunoh et al.2000), kanamycin, gentamicin, and streptomycin (Madigan et al.2003); enzymes such as laminarase, cellulose, and chitinase (Crawford et al.1993); anticancer drugs, immunosuppressors and enzyme inhibitors (Hwang et al.2007) are of considerable importance in industry (Seong et al. 2001) as they represent a large part of the rhizosphere microbial flora (Sardi et al.1992). It has also been demonstrated that they are important both qualitatively and quantitatively in rhizosphere where they may influence plant growth and protect plant roots against invasion of root pathogenic fungi (Crawford et al. 1993; Cao et al. 2004). The use of actinomycetes as excellent biological control agents of soil-borne root disease is of interest. Recent study shows the potential of actinomycetes as root colonizers and fungal antagonists (Kunoh et al. 2000), in which these group are highlighted as interesting biological control agents.

Therefore, the primary objective of the present invention is to overcome the aforementioned limitations associated with these conventional methods by providing an environmental friendly approach to control the Ganoderma infection in oil palm.

Besides that, another objective of the present invention is to provide a cost-effective method in protecting oil palm from Ganoderma boninense infection.

In addition, present invention aims to provide a mass- producible biological control agent (BCA) that is easy to use and do not require much skill or technique in applying. Moreover, present invention solves the problem of storage, transport and handling BCA.

It is yet another object of the present invention to provide a composition comprising a BCA that is effective in suppressing the growth of causative pathogen of BSR disease in oil palm.

SUM ARY OF INVENTION

In one aspect of the present invention, a composition, method of preparation of said composition and method of biologically controlling Ganoderma infection in plants, particularly basal stem rot disease primarily caused by Ganoderma species in oil palm is disclosed.

The composition comprising Streptomyces species, the actinomycetes bacteria isolated from the soil of unaffected healthy oil palm. Microbial carrier of vermiculite is added into said composition to stabilize and maintain viability of the bacterial cell for a long period of time, suitably for long-term storage.

The method of preparing biological controlling agent (BCA) comprising the steps of isolating Streptomyces bacteria from soil of unaffected, healthy oil palm, selecting a single colony of bacteria culture, culturing said bacteria in liquid media, preparing said bacteria into cell suspension, homogenizing cell suspension to break up the mycelia into small fragments and mixing said cell suspension with microbial carrier. The microbial carrier is vermiculite .

The method for reducing susceptibility of a plant to fungal infection, comprising the steps of providing a composition comprising substantially bacterial suspension of

Streptomyces nigrogriseolus GanoSAl isolated from soil of unaffected healthy oil palm plantation, and vermiculite; and allowing Streptomyces nigrogriseolus GanoSAl to proliferate and protecting plant from fungal infection by applying the composition directly onto the soil where the plant to be treated will grow and uprooting seedlings and adding the composition into the soil.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the percentage of dead seedlings (DS) , disease incidence (DI) and severity of foliar symptoms (SFS) of oil palm seedlings after infected with G. boninense for six months.

DETAILED DESCRIPTION OF THE INVENTION

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.

The composition according to a preferred embodiment of the present invention may be prepared based on a pure culture of actinomycetes bacteria which serves as biological control agent (BCA) to restrict growth of Ganoderma species, in particular G. boninense that leads to developing basal stem rot (BSR) disease. The composition may be in the form of, but not limiting to powder. In one preferred embodiment, the BCA is Streptomyces, and more preferably species Streptomyces nigrogriseolus . The actinomycetes are isolated by present inventor from soil of unaffected, healthy oil palm plantation in Kluang, Malaysia. The Streptomyces strain isolated is Streptomyces nigrogriseolus GanoSAl. Bacterial suspension of isolated strains of Streptomyces nigrogriseolus GanoSAl may be directly introduced into the soil by various methods such as spraying, irrigation, etc. However, bacterial cells suspension is easily drained-off when it applies around the palm of field and it may not be as effective as utilizing a host. The present invention provides method for controlling G. boninense using the Streptomyces GanoSAl powder, incorporated with the Streptomyces GanoSAl culture in optimized medium. In accordance to the preferred embodiment of the present invention, there is disclosed a method for producing the formulated forms of the actinomycetes as identified above.

The present disclosure of the present invention provides actinomycetes bacteria that have been identified to exhibit substantial effects in controlling the BSR infection in oil palm. It is observed that the composition comprising said bacteria showed significant reduction of Disease Incidence (DI, %) in germinated seedlings of oil palm, lower Dead Seedlings (DS, %) occurrence and reduced Severity of Foliar Symptoms (SFS, %) .

The present disclosure further provides the isolation of bacteria identified as Streptomyces GanoSAl, whereby said bacteria has unusual effects against BSR, and having unique inoculants feature in controlling Ganoderma disease of oil palm.

In one embodiment, the composition of present invention comprises of microbial carrier in powder form whereby it improves aeration and moisture retention. The carriers are provided to stabilize and maintain viability of BCA in composition. The porous nature of such carrier enables holding of microbial cells which relate to slow release of the BCA incorporated directly into or surround the soil.

Suitable microbial carriers employed in the present invention include light mica-like minerals, for example, vermiculite, kaolin, alginate, talc, clay, fir bark, perlite and silica. The preferred embodiment of carrier is vermiculite due of its excellent pH buffering capacity, and able to protect the microorganism from environmental stresses . As understood, the composition can be applied to plants by soil inoculation or by dipping the plant roots in the BCA composition before planting. However, seed coating method is preferred, and it is recognized as the most effective way for the Streptomyces GanoSAl in the BCA composition to colonize the roots of the plants during seed germination.

Despite the fact that the active ingredient of the composition is the strain of Streptomyces nigrogriseolus GanoSAl isolated from oil palm, and apparently, is an excellent fungicide for oil palms, however, it may also be applied to various types of plants includes vegetables, ornamental crops, fruit crops and plantation crops in controlling Ganoderma-related infection. 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 14 000022

11 characteristics and embodiments of this invention, therefore various changes may be provided to adapt to various usages and conditions.

Example 1

Characterization of Streptomyces spp. GanoSAl

A standard sequence analysis was performed to identify the DNA profile of the actinomycetes used in accordance with the present invention. As briefly discussed above, it is observed that the profile of the Streptomyces GanoSAl is predicted as putative Streptomyces nigrogriseolus with 99% of identity. Using sequences from National Center for Biotechnology Information (NCBI) database, alignment had been done between Streptomyces GanoSAl and Streptomyces nigrogriseolus . The sequences were aligned at 10 base pairs (bp) to 1000 bp on Streptomyces nigrogriseolus sequence.

The sequence obtained is as follows:

AATCGGCGGCCAAACCTTCGGGGTGGATCAGTGGCGAACGGGTGAGTAACACGTGGGCA ATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATATGACCTT CCTCCGCATGGGGGCTGGTGTAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCA GCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCG ACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAA TATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCG GGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCA CCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAAT TATTGGGCGTAAAGAGCTCGTAGGCGGCCTGTCGCGTCGGATGTGAAAGCCCGGGGCTT AACCCCGGGTCTGCATTCGATACCGGCAGGCTAGAGTGTGGTAGGGGAGATCGGAATTC CTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAAGCGGATCT CTGGGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAAGATTAGATACCC TGGGAGTCCACGCCGTAAACGTTGGGAACTAAGTGTTGGGGGACATTCCCCGTCGTCGG TGCCGCGGCTAACACATTAAATT

Example 2

Preparation of BCA Composition

The composition 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.

A single colony of fresh culture Stre tomyces nigrogriseolus GanoSAl isolated from soil of healthy palm plantation in Kluang, Malaysia was inoculated into 10 mL yeast extract-malt extract broth to prepare seed culture. The actinomycetes culture is then prepared by inoculating 10 mL of seed culture in 100 mL of liquid media. In one preferred embodiment, the liquid media comprises yeast extract, malt extract and dextrose at pH 7. The flask containing cell suspension was then placed on an orbital shaker, which operated at a speed of approximately 150 rotations per minute (rpm) for around 7 days, at temperature of approximately 28 °C. Soon after 7 days of incubation, cell suspension was homogenized using medium speed of homogenizer to break up the mycelia into small fragments. The homogenate cell suspension was subjected to dilution for colony count. The living culture was then incorporated into microbial carrier to prepare the BCA powder formulation. This was initiated by preparing a sterile delivery medium. In one preferred embodiment, delivery medium used was vermiculite. The delivery medium was sterilized by autoclaving twice at 121°C for about 30 minutes and drying by heat at approximately 60°C for 2days. A standardized actinomycetes culture containing 10⁸ CFU/mL was then prepared. The BCA composition was prepared by incorporating about 20mL of the culture prepared earlier into 50g of microbial carrier in sterile condition. The BCA composition was then packed and sealed in a polyethylene bag. The packages were stored in dry condition at room temperature (25 ± 2°C) to ensure BCA cell viability at a substantially high level over a long period of time. The composition as described above was tested on seedlings of oil palm, whereby said seedlings were treated with the powder of the present invention and inoculated with G. boninense . The composition was applied periodically based on the growth age of the oil palm.

Example 3

Effectiveness of the BCA Composition against Ganoderma boninense

A field evaluation was performed to determine antagonistic effect of the BCA composition of the present invention against Ganoderma infection in oil palm. Said field evaluation study was conducted in oil palm nursery at MPOB- UKM Research Station, Bangi, Selangor, Malaysia.

The procedures for use in the event of field evaluation of the preferred embodiment are as mentioned as follows:

The planting material used was Dura x Pisifera (D x P) at 4 month-old oil palm seedlings. The oil palm seedlings were maintained in the nursery for a month prior to the treatment. The seedlings were pre-treated with respective treatments by adding 50g of the BCA formulation in the soil. 14 days after initial inoculation as described earlier, the seedlings were then uprooted carefully and 50g of the BCA formulation was added into the soil as a booster inoculation. Germinated seeds containing composition without BCA cell suspensions added were used as control subjects (untreated seedlings) . The treated seedlings were then inoculated with G. boninense using the rubber wood block (RWB) sitting technique as described by Idris et al. (2006) .

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. All of the oil palm seedlings were arranged in Randomized Complete Block Design (RCDB) . Each trial was conducted with two treatments and three replicated with six seedlings per replicate. The two treatments were as listed follows:

CI: Seedlings untreated with Streptomyces GanoSAl and inoculated with G. boninense

Tl: Seedlings treated with Streptomyces GanoSAl and inoculated with G. boninense

The effectiveness of composition in controlling BSR disease development in oil palm seedlings was evaluated based on quantitative assessment measured as percentage of disease incidence (DI), severity of foliar symptoms (SFS) , dead seedlings (DS) and disease reduction (DR) at monthly intervals .

Figure 1 shows the quantitative assessment of BCA composition enriched with Streptomyces GanoSAl in controlling BSR disease caused by G. boninense after six months of treatment. Seedlings treated with the BCA composition (labeled as b) were less susceptible to the BSR disease (53.3% DI) as compared with the untreated seedlings (labeled as a) (93.3% DI) . Furthermore, it was showed that SFS in seedlings treated with the bio-control agent composition was significantly lower (49.68% SFS) than that of the untreated seedlings which had caused 83.84% of SFS. A significant low percentage of dead seedlings (40.00% DS) was also recorded in seedlings treated with the bio-control agent composition as compared to the untreated seedlings with 86.67% DS . The results showed the means with two sets of treatment are significantly different according to t- test at p<0.05. Table 1: The Effect of the BCA composition on BSR Disease Development in Oil Palm Seedlings after 6 Months of Treatment

In reference to Table 1 as shown above, the seedlings treated with Streptomyces GanoSAl give the lowest Area Under Disease Progression Curve (AUDPC) of 109.99 compared to untreated of 273.33. The Ganoderma infection in treated oil palm was reduced (Disease Reduction, DR) about 59.76% as compared to untreated palms. It can be concluded that the control composition GanoSAl is efficient in controlling Ganoderma infection in oil palm seedlings.

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 a variety of 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.

Claims

1. A composition for biologically controlling fungal infection in plants comprising:

at least one species of actinomycetes bacteria; and

at least one type of microbial carrier to allow binding of said actinomycetes bacteria.

2. The composition as claimed in claim 1, wherein said fungal infection is caused by Ganoderma species, wherein it is further characterized by Ganoderma boninense .

3. The composition as claimed in claim 1, wherein said fungal infection causes basal stem rot (BSR) disease.

4. The composition as claimed in claim 1, wherein said plants is oil palm.

5. The composition as claimed in claim 1, wherein said actinomycetes bacteria is Streptomyces species, wherein it is further characterized by Streptomyces nigrogriseolus GanoSAl .

6. The composition as claimed in claim 5, wherein said Streptomyces nigrogriseolus GanoSAl is isolated from soil of unaffected, healthy oil palm.

7. The composition as claimed in claim 1, wherein said microbial carrier is light mica-like minerals selected from a group of comprising vermiculite, kaolin, algimate, talc, clay, fir bark, perlit, silica, or combination thereof.

8. The composition as claimed in claim 7, wherein said microbial carrier is vermiculite.

9. The composition as claimed in claim 1, wherein said composition is in powder form.

10. A method of preparing biological controlling agent (BCA) comprising the steps of:

isolating actinomycetes bacteria from soil of unaffected, healthy oil palm;

culturing said bacteria in liquid media;

preparing said bacteria into cell suspension; and

mixing said cell suspension with microbial carrier.

11. The method as claimed in claim 10, wherein said actinomycetes bacteria is Streptomyces species, wherein it is further characterized by Streptomyces nigrogriseolus GanoSAl .

12. The method as claimed in claim 10, wherein said step of isolating further comprising the step of selecting a single colony of bacteria culture.

13. The method as claimed in claim 10, wherein said step of preparing said bacteria into cell suspension further comprising the step of homogenizing cell suspension to break up the mycelia into small fragments.

14. The method as claimed in claim 10, wherein said microbial carrier is light mica-like minerals selected from a group of comprising vermiculite, kaolin, algimate, talc, clay, fir bark, perlit, silica, or combination thereof.

15. The method as claimed in claim 11, wherein said microbial carrier is vermiculite.

16. A method for reducing susceptibility of a plant to fungal infection, comprising the steps of:

providing a composition comprising substantially bacterial suspension of Streptomyces nigrogriseolus GanoSAl isolated from soil of unaffected healthy oil palm plantation, and vermiculite; and

allowing Streptomyces nigrogriseolus GanoSAl to proliferate and protecting plant from fungal infection.

17. The method as claimed in claim 16, wherein said steps of allowing Streptomyces nigrogriseolus GanoSAl to proliferate further comprising the step of applying the composition directly onto the soil where the plant to be treated will grow.

18. The method as claimed in claim 16, wherein said steps of allowing Streptomyces nigrogriseolus GanoSAl to proliferate further comprising the step of uprooting seedlings and adding the composition into the soil.