The Potential of Streptomyces as Biocontrol Agents against the Rice Blast Fungus, Magnaporthe oryzae (Pyricularia oryzae)

1Rice (Oryza sativa) is an important food crop and it is the staple diet of over three billion people around the world, particularly in Asia1. Nearly half of Asia’s population depends on rice as their main food source, making Asia the region with the highest rice consumption—more than 110 kg per capita annually. Rice is grown in more than a 100 countries across a wide range of climatic conditions, ranging from rivers deltas to mountainous regions. Asian countries including China, India, Indonesia, Thailand, Philippines, Vietnam, Bangladesh, and Myanmar account for approximately 90% of the world’s total rice production. The other rice-producing countries include Egypt, Brazil, sub-Saharan countries, and the USA. Wherever it is grown, however, rice is susceptible to diseases with far reaching economic implications. Infection with phytopathogenic fungi are among the most worrying of these diseases as it may result in significant crop yield losses, and additionally, some of the fungi produce compounds which are potentially toxic upon consumption. For instance, several Fusarium, Aspergillus, and Penicillium species are capable of producing mycotoxins (e.g., aflatoxins, citrinin, fumonisins, ochratoxin A, and zearalenone) which can be harmful to human beings if they are ingested via consumption of contaminated rice. Given that the present methods of preventing rice diseases are not entirely satisfactory from several angles, it is imperative to seek new and effective methods of prevention in order to produce rice that is safe for consumption as well as to reduce crop yield losses.

2Currently, Magnaporthe oryzae (anamorph Pyricularia oryzae) is regarded as one of the most important phytopathogenic fungi as it is the causal agent of rice blast— the most destructive disease of rice. M. oryzae B. Couch (anamorph Pyricularia oryzae Cav.), also known as Magnaporthe grisea (Hebert) Barr (anamorph Pyricularia grisea Sacc.), is a haploid filamentous ascomycete fungus. M. oryzae is defined as a new species distinct from M. grisea based on multilocus gene genealogy and laboratory mating experiments by. On the basis of phylogenetic analysis, Magnaporthe is now separated into two distinct clades—one clade associated with Digitaria (crabgrass) maintains the name M. grisea, while the other clade associated with rice and other cultivated grasses was characterized as a novel species and given the name M. oryzae. Given the phylogenetic differences, however, there are no morphological differences between the isolates from these two clades. As a result, the names M. oryzae and M. grisea are still used interchangeably by scientists for the fungal isolates that infect rice.

3Magnaporthe oryzae infects the aerial parts of the rice plant — including leaves, nodes, stems, and panicles— at all stages of development. Infection results in rice blast symptoms such as leaf blast, node blast, collar rot, neck rot, and panicle blast; this generally manifests as purplish/grayish/brownish/whitish spots or lesions as well as withering of leaves. This fungus was later discovered to also have the ability to infect the roots of the rice plant; and infection of the root may eventually spread to the aerial tissues, causing rice blast disease. However, the exact nature and sequence of the process by which rice blast infects the roots of the rice plant has yet to be fully eludicated. Rice blast disease has been reported in approximately 85 countries, mainly in Asia, Africa, and Latin America. Yield loss due to rice blast ranges from approximately 10–30% annually in the various rice producing countries and can reach up to 50% during disease epidemics.

4Efforts have been made by researchers to identify and analyze the avirulence (AVR) genes of M. oryzae. This would serve as the basis of understanding fungal mechanisms of pathogenesis and clarifying the mechanisms responsible for the coevolution of fungal effectors and their host targets. Besides this, development of cultivar-specific resistance involving gene-for-gene system can be achieved by conditioning resistance through high-yielding rice cultivars carrying single dominant disease resistance (R) genes to a single corresponding dominant AVR gene in a particular pathogen strain. To date, four AVR genes have been isolated from M. oryzae: PWL1 and PWL2 genes which encode Gly-rich hydrophobic proteins with secretion signal sequences, AVR-Pita gene which encodes a putative secreted protein with similarity to metalloproteases, and ACE1 gene which encodes a putative hybrid protein of a polyketide synthase and a peptide synthase. In contrast, more than 25 resistance (R) genes encoding proteins that recognize M. oryzae AVRs have been mapped on the rice genome.

5Besides breeding of cultivar-specific resistance, several control methods have been attempted to manage plant diseases. Among these, chemical control is the most commonly used method yielding effective management of plant diseases. With reference to control of rice blast, chemical control involves the use of pesticides, specifically fungicides. A number of fungicides have been used against this disease, for instance, azoxystrobin, benomyl, carbendazim, carpropamid, dithiocarbamate, edifenphose, fenoxanil, tiadinil, tricyclazole, pyroquilon, probenazole, iprobenfos, isoprothiolane, metominostrobin, and propiconazole. Generally, the effectiveness of fungicides depends on several factors: the compound itself, the timing and method of application, the level of disease present, the efficiency of disease forecasting systems, and the rate of emergence of fungicide resistant strains. Although they are effective at controlling the fungal infections in rice, there are growing public concerns over the use of synthetic fungicides. Misuse and excessive use of synthetic pesticides (e.g., fungicides, insecticides, and herbicides) might cause environmental pollution, residual toxicity, development of pesticide resistance, reduce soil quality, and damage to natural ecosystems. Furthermore, human exposure to pesticides may cause poisoning and harmful side-effects to organs and/or biological processes. Pesticide poisoning is a significant occupational health issue in developing countries, likely due to insufficient or poor occupational safety practices. Appropriate work practices are essential to ensure the safety of workers, particularly in the case of agricultural workers who are often exposed to pesticides during application and handling operations such as mixing, cleaning, loading spray equipment, and disposing of empty containers. Side effects and symptoms caused by exposure to pesticides have been reported in several developing countries. For instance, farmers in Vietnam reported symptoms such as skin irritation, headache, dizziness, eye irritation, shortness of breath, and acetyl cholinesterase inhibition due to the exposure to various pesticides during mixing and spraying.

6New and improved fungicides with minimal side effects are required in order to prevent these concerns. Nowadays, natural products which are safe for the environment and have low toxicity to living organisms are gaining interest as important sources for the development of fungicides, and these may serve as effective substitutes for synthetic fungicides. Moreover, another alternative approach to the use of fungicides is the use of microbial antagonists as biocontrol agents. Biocontrol agents are microorganisms that suppress plant pathogens; they can achieve biological control through competition, antibiosis, and hyperparasitism. Biological control of plant diseases is known to be more cost effective, safe and environmentally friendly as compared to the use of fungicides. Streptomyces bacteria are among the microbial antagonists that have been exploited for the biological control of plant diseases. This review aims to encapsulate the current body of knowledge of the biocontrol potential of Streptomyces against the rice blast fungus, M. oryzae.

7Streptomyces is the largest genus of the phylum Actinobacteria and was first proposed by. The genus Streptomyces consists of a group of Gram-positive, aerobic, non-motile, catalase positive, and non-acid-fast bacteria with a filamentous form that resembles fungi. Currently, over 700 species of Streptomyces have been identified2 and these bacteria have relatively large genomes of approximately 8–9 Mbp in size with a high GC content of more than 70%.

8The members of Streptomyces are well-known for their ability to produce a variety of bioactive compounds with different bioactivities such as antibacterial, antifungal, antiviral, immunosuppressive, anticancer, and antioxidant properties. These bioactive compounds have important applications in various fields. For example, approximately 75% of commercially useful antibiotics were derived from the genus Streptomyces and they are thus the primary antibiotic-producing organisms exploited by the pharmaceutical industry. Streptomyces strains also have important applications in the agricultural field through their biological control potential against phytopathogens, particularly phytopathogenic fungi. In line with this, research on the biological control of rice blast disease using different Streptomyces species has been conducted under laboratory conditions, as well as in greenhouse or growth chamber conditions.

9Studies have reported in vitro antagonism of different Streptomyces spp. against M. oryzae which were tested using the dual culture method. Dual culture method has been widely used for preliminary screening of biocontrol agents including fungi and bacteria. This method allows the biocontrol agent and pathogen to interact on a solid medium in a Petri dish, under optimal conditions for both organisms. The degree of inhibition is recorded by observing the inhibition zone produced or the overgrowth of the pathogen by the biocontrol agent.

10In a study conducted by, S. vinaceusdrappus isolated from Loktak lake sediment was first reported to exhibit antagonistic activity against M. oryzae (P. oryzae MTCC 1477). S. vinaceusdrappus also showed maximal antagonistic activity against most of the rice fungal pathogens tested, which include Curvularia oryzae, Bipolaris oryzae, and Fusarium oxysporum. S. vinaceusdrappus inhibited the mycelial growth of P. oryzae by 53.5%, which was relatively good since more than 50% of the mycelial growth was inhibited. reported that Streptomyces philanthi RM-1-138 isolated from rhizosphere soil of chili pepper in Southern Thailand exhibited significant antifungal activity against M. oryzae (P. oryzae PTRRC-18), with 88.73% inhibition of mycelial growth of the rice blast fungus. This suggests that S. philanthi RM-1-138 has greater inhibition against the rice blast fungus as compared to S. vinaceusdrappus.

11The in vitro assay conducted by showed that Streptomyces globisporus JK-1 demonstrated the most pronounced inhibitory effects against M. oryzae as compared to other phytopathogenic fungi tested in the study. S. globisporus JK-1 inhibited mycelial growth of M. oryzae with an inhibition zone of 15 mm out of 35 mm. A study also showed that endophytic Streptomyces from rice cultivars in China demonstrate antagonism against rice fungal pathogens, particularly 54.5% of Streptomyces griseofuscus and 21.8% of Streptomyces hygroscopicus were the most active among the studied population of antagonistic endophytic Streptomyces which exhibited strong antagonism against M. oryzae. Streptomyces sindeneusis isolate 263 and Streptomyces isolate 339 obtained from agricultural soils of Kerman in Iran were also found to inhibit M. oryzae. In addition, reported that Streptomyces flavotricini isolated from Egyptian rice field soils showed the strongest antifungal activity against M. oryzae; the antifungal compound produced by S. flavotricini was successfully purified and identified as dihydroxy viridiofungin (C37H58N2O10).

12Based on these findings, S. vinaceusdrappus, S. philanthi RM-1-138, S. griseofuscus, S. hygroscopicus, Streptomyces isolate 339, and S. flavotricini are potential candidates for use as biocontrol agents against rice blast as they possess inhibitory activity against M. oryzae (Table 1). Based on the percentage of mycelial growth inhibition, S. philanthi RM-1-138 appears to be one of the most promising agents for the inhibition of M. oryzae. However, studies involving greenhouse or field experiments are still required to more definitively evaluate the biocontrol potential of these Streptomyces spp. against M. oryzae.

13The limitation of laboratory experiments is that they only prove the antagonistic activity of Streptomyces spp. against M. oryzae under certain conditions. The antagonism exhibited by Streptomyces in laboratory experiments might not necessarily reflect antagonism under greenhouse or field experiments. Greenhouse or growth chamber experiments are conducted for the purpose of further evaluating the efficacy of Streptomyces strains as biocontrol agents. For S. sindeneusis isolate 263 and S. globisporus JK-1, studies on their biocontrol potential against the fungus under greenhouse conditions were conducted by and respectively. reported that typical blast symptoms were observed when rice plants at the three leaf-stage of vegetative phase were treated with M. oryzae. The percentage of diseased leaf area was evaluated according to the method developed by the International Rice Research Institute (IRRI). It was found that the rice plants receiving treatment with S. sindeneusis isolate 263 showed significantly reduced lesion development. The diseased leaf area was 8% for rice plants treated with M. oryzae alone, while it was only 0.5% for rice plants treated with S. sindeneusis isolate 263 in combination with M. oryzae. This result indicates that S. sindeneusis isolate 263 acted as an antagonist.

14compared the control of the rice blast using Streptomyces and fungicide by infecting rice plants at the five leaf-stage during the vegetative growth phase with M. oryzae, followed by treatment with culture filtrates of S. globisporus JK-1 and tricyclazole respectively. Tricyclazole is one of the commonly used fungicides for the control of rice blast disease with several advantages over other fungicides, for instance, it is systemic in rice for blast control and has long residual effectiveness. The results showed that control efficacy for S. globisporus JK-1 treatment was 88.3% and for tricyclazole was 79.4%, compared to the inoculated control. This suggests that S. globisporus is as efficient and possibly even superior to tricyclazole, with the additional benefits of biocontrol agents as compared to synthetic agents described earlier.

15Additionally, a novel Streptomyces strain, BG2-53, with 96% homology to S. lipmanii based on analysis of 16S rDNA sequences, exhibited potent antifungal activity against M. oryzae under growth chamber conditions. The strain showed the highest degree of fungal control in comparison to fungicides such as Blasticidin-S and Tricyclazole. However, the evaluation on the extent of rice disease infection was solely based on visual estimation, unlike other studies where the Standard Evaluation System of IRRI was applied.

16Overall, the results of greenhouse and growth chamber experiments strongly suggest that several Streptomyces spp. possess antagonistic activities against M. oryzae, and therefore have the potential to effectively control rice blast. Field experiments are still required to more definitively estimate the efficacy of Streptomyces spp. as biocontrol agents under real life conditions as environmental factors greatly affect their performance.

17The suppression of rice blast in the greenhouse by certain Streptomyces might indicate the presence of bioactive compound(s) with antifungal activity against M. oryzae in the culture filtrates. Streptomyces are prolific producers of bioactive compounds. Some well-known antibiotics produced by Streptomyces have been used as fungicides for the control of rice blast, for instance, Blasticidin-S and Kasugamycin. Blasticidin-S, isolated from S. griseochromogenes was the first antibiotic commercially introduced for the control of rice blast in Japan. Kasugamycin was discovered soon after; it was first isolated from S. kasugaensis by. Kasugamycin has been safely used to protect rice plants against blast disease; it has relatively low mammalian toxicity and no phytotoxicity toward rice plants and most crops. Recently, the significance of Blasticidin-S as a fungicide has decreased as it has been replaced by new pathogen-specific fungicides with lower toxicity. Kasugamycin is currently still on the market and is sold in several different formulations such as wettable powder, granule, and soluble liquid under the trade names Kasumin and Kasumin-Bordeaux from Hokko Chemical Industry, Co., Ltd.

18Several studies have performed testing of the various compounds produced by Streptomyces on M. oryzae. These studies have shown that several compounds produced by Streptomyces spp. exhibited antifungal activity against M. oryzae (Appendix 1). The antibiotic Oligomycin A was first isolated from S. diastatochromogenes and was found to be active against several other phytopathogenic fungi in addition to M. oryzae such as Botrytis cinerea, Cladosporium cucumerinum, Colletotrichum lagenarium, Phytophthora capsici, Alternaria alternata, and Aspergillus niger. Oligomycin A’s ability to control the development of rice blast was evaluated in the greenhouse and the results showed that rice plants treated with Oligomycin A (50 μg/mL) had reduced lesions. When the concentration of Oligomycin A was increased up to 500 μg/mL, the rice plants did not show any rice blast disease symptoms.

19Rapamycin—also known as Sirolimus—was initially isolated from S. hygroscopicus. Rapamycin is a potent antifungal agent found to be effective against Candida albicans, Microsporum gypseum, and Trichophyton granulosum. reported that Rapamycin inhibited the growth of Mucor circinelloides and Rhizopus oryzae, both of which are post-harvest fruit pathogens.

20Other compounds such as Pyrroles (Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-) are commonly found in various Streptomyces species. Furthermore, Pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(phenylmethyl) was found to be able to protect plants from phytopathogenic fungi.

21Bioactive compounds derived from Streptomyces can be used for the management of fungal plant diseases as an alternative to synthetic fungicides. These natural compounds are biologically synthesized and often biodegradable. Thus, they may be useful for the development of agricultural fungicides that are more pathogen-specific with minimal side-effects toward the environment. In order to formally establish any organism as a commercially viable biocontrol agent, studies involving the application of the microbial antagonist in question on a certain plant for the control of a particular pathogen are required to investigate their effectiveness. Although it is too early to draw any definite conclusions, the Streptomyces spp. listed in Table 1 could be considered potential biocontrol agents, as they are capable of producing compounds with antifungal activity against M. oryzae.

22It is known that antagonistic activities of bacteria against fungal pathogens can be achieved through three main mechanisms: competition for nutrients and space, antibiosis, and parasitism. The advantages of Streptomyces spp. include their ability to colonize plant root surfaces, survive in various types of soil and also produce spores which allow them to survive longer and in various extreme conditions. Antibiosis happens when the antagonist present in the plant produces metabolites such as antibiotics or antifungals which can inhibit or kill the pathogen. Streptomyces spp. used as biocontrol agents produced antibiotics such as geldanamycin and nigericin for the control of plant diseases, which were proven by the presence of antibiotics in soil. Likewise, hyperparasitism may occur due to the release of extracellular lytic enzymes such as chitinases and glucanases from the biocontrol agent. It has also been shown that Streptomyces spp. are capable of producing chitinases and glucanases which play important roles in destruction of fungal cell walls. The colonization ability and competitive traits of Streptomyces could result in successful competition against phytopathogenic fungi and suppression of their growth. It can be presumed that the suppression of rice blast by Streptomyces spp. might be due to these mechanisms, but further studies are required to provide evidence regarding the actual biocontrol mechanisms of Streptomyces against M. oryzae.

23One of the major challenges encountered during the selection of biocontrol agents is that biocontrol agents that appear efficacious based on in vitro experiments might not be effective in controlling plant diseases in greenhouse or field conditions. The efficacy of biocontrol agents is affected by organic matter, pH, nutrient level, and moisture level of the soil. Owing to the variations in environmental conditions in different locations, biocontrol agents that perform well in in vitro conditions might fail in greenhouse or field experiments. Therefore, the environmental factors at the location where biocontrol agents will be applied should be taken into consideration during the selection of appropriate biocontrol agents. Ideally, the biocontrol agents should be isolated from and applied to locations with similar environmental factors in order to achieve successful biological control. Furthermore, the formulation (e.g., powder, liquid, or granule) and the method of application of biocontrol agents such as soil inoculation, seed inoculation, and vegetative part inoculation should be examined (Figure 1) as they are important in determining the outcomes of field experiments. Soil inoculation involves mixing of the biocontrol agent with soil or spreading the biocontrol agent in sowing furrows by drip systems. Seed inoculation involves dipping seeds in a culture of the biocontrol agent or mixing the seeds with the inoculant using appropriate wetting agents. Vegetative part inoculation involves aerial/foliar spraying of the biocontrol agent or seedling treatment by dipping the roots of the seedlings into a solution containing the biocontrol agent prior to transplantation (Figure 1). The appropriate application method is likely to contribute significantly to the success of the biocontrol agents in the field trials.

24Some Streptomyces spp. have been successfully developed into commercial biocontrol agents and tested for the control of other plant diseases. For example, Streptomyces griseoviridis strain K61 (Mycostop®), which has been tested for the control of Ceratocystis radicicola that causes black scorch on date palm and soilborne pathogens of tomato such as Fusarium oxysporum f.sp. lycopersici and Verticillium dahliae. Other commercial Streptomyces biocontrol agents include S. lydicus WYEC108 (Actinovate®, Micro108®, Action Iron®), and S. saraceticus KH400 (YAN TEN S. saraceticus). Biocontrol agents are relatively safe toward humans as no adverse effects in users and other workers have been reported following exposure to these commercial products. However, hypersensitivity may occur in certain individuals on exposure to biological dust produced during handling of S. griseoviridis strain K61 (Mycostop®) dry end-product. Hence, wearing appropriate safety equipment is required when handling these agents. With reference to the control of rice blast disease, however, commercial Streptomyces biocontrol agents have yet to be developed.

25Rice blast, the result of infection by M. oryzae, is the most destructive disease of rice, leading to crop yield losses and economic damage. While chemical control has been the mainstay of controlling this infection, biological control has now been introduced as an alternative for the management of rice blast disease. Biological control of plant diseases is typically inexpensive, long lasting, and safe toward the environment and living organisms; however, biological control can be a slow process and the search for suitable biocontrol agents requires considerable time and effort. Streptomyces spp. certainly demonstrate the potential to be developed as biocontrol agents due to their various beneficial properties. Based on current research findings, S. vinaceusdrappus, S. philanthi RM-1-138, S. griseofuscus, S. hygroscopicus, Streptomyces isolate 339, and S. flavotricini showed antifungal activity against M. oryzae under in vitro conditions. S. sindeneusis isolate 263 and S. globisporus JK-1 demonstrated in vitro antifungal activity against M. oryzae as well as successful biocontrol of rice blast in greenhouse experiments. Streptomyces strain BG2-53, which appears to be a novel strain, showed antifungal activity against M. oryzae under growth chamber conditions. These Streptomyces spp. possess antagonistic activities against M. oryzae, with S. globisporus JK-1 showing high control efficacy of up to 88.3%. Furthermore, studies have revealed that Streptomyces produces various compounds with antifungal activity against M. oryzae. Therefore, they are excellent candidates as biocontrol agents for the biological control of this devastating rice blast disease. In order to establish Streptomyces as biocontrol agents, more field experiments should be conducted to determine their control efficacy under different environmental conditions. Additionally, more work is needed to optimize isolation, formulation and application methods of Streptomyces in order to fully maximize their potential as effective agents to control rice blast.

26The literature review and manuscript writing were performed by JL and H-LS, while TK, L-HC, PP, K-GC, B-HG, and L-HL provided vital guidance and insight for the writing. The research topic was conceptualized by L-HL and B-HG.

27The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.