Full Length Research Paper
ABSTRACT
Pyricularia oryzea (Magnaporthe oryzae) causes blast diseases in rice (Oryza sativa) in Mali. The losses could reach 90% of production during rainy weather conditions. Isolation and characterization of M. oryzae and Trichoderma species were carried out to assess the importance and distribution of the pathogen and antagonist Trichoderma species in rice fields in Sikasso (Mali), and select, in vitro, Trichoderma species with high pathogen biocontrol activity. In the pathogen isolation, only one isolate of M. oryzae were obtained, while 12 Trichoderma isolates were obtained. In the fungal growth tests three isolates of Trichoderma: Trichoderma harzianum S31, T. harzianum S32, and T. harzianum S33 highly inhibited the growth of the pathogen with a coefficient of antagonism of 0.55, 0.71 and 0.78 respectively. These isolates were selected for further greenhouse and field tests.
Keys words: Rice blast disease, Trichoderma, Magnaporthe oryzae, Pyricularia oryzea, antagonism, Oryza sativa, Mali.
INTRODUCTION
Rice (Oryzae sativa) is a staple food and cereal crop for more than half of the population in Mali where agriculture drives the national economy (ZEF, FARA, IER (2017)) Mali is one of the top rice producers in West Africa with a 3.19 million tons of rice produce in 2019 (FAOSTAT, 2019). Unfortunately, the country is also particularly vulnerable to agricultural diseases (Gurr et al., 2011), mainly rice blast diseases, which limit rice yields to below the global average, threatening smallholder farmers’ livelihoods as well as food and economic security (USDA, 2012; Asibi et al., 2019; Soullier et al., 2020).
The fungal plant pathogen Magnaporthe oryzae, involved in causing serious blast diseases in rice in Mali, is very difficult to manage. At present, the losses caused by this pathogen could reach 30 to 90% of the rice production in the affected areas (Savary et al., 2000; Khemmuk, 2016). For that, chemicals, compounds are widely used to control blast disease pathogens (Dougoud et al., 2018). Pyricularia oryzae (M. oryzea) is the causal agent of this disease on wheat (Tembo et al., 2020), maize (Pordel et al., 2021) and Panicum repens (Well et al., 2005). It can cause blast disease on the three hosts. Pordel et al. (2021) in pathogenicity assays in greenhouse revealed that strains from maize can infect barnyard grass and conversely.
Biological control is a promising tool to maintain good level of agricultural production while reducing the release of polluting chemical pesticides to the environment. Many researches showed that soil microorganisms, mainly fungi species, are promising as biocontrol agents (Swain et al., 2018; Sood et al., 2020; Es-Soufi et al., 2020). Out of these microorganisms, Trichoderma species, filamentous fungi previously considered to be culture contaminants species, are common inhabitant of rhizosphere and contribute to control of many soil-borne plant-diseases caused by fungi (Chuaki et al., 2002; Bastakoti et al., 2017). Trichoderma spp., have been largely studied as biological control agents against plant pathogens (Rivera-Méndez et al., 2020; Alfiky and Weisskopf, 2021; Bastakoti et al., 2017). In recent years, considerable success has been achieved in the control of plant diseases by the use of Trichoderma isolates which have become commercially available as biocontrol agents (Woo et al., 2014; Es-Soufi et al., 2020). Chou et al. (2019) combined the use of Trichoderma harzianum and a resistant rice variety, considered as sustainable approaches to reduce yield losses and to cope with recent restrictions on fungicide use to manage blast disease, showed that T. harzianum reduced the incidence of leaf blast and neck blast on IR504 (susceptible strain), but its efficacy was not consistent and the magnitude of disease suppression by T. harzianum was higher for neck blast than for leaf blast. Also, Mouria et al. (2018) applying T. harzianum at a concentration of 108 spores/ml, in alternation with the mancozeb at 1000 ppm against rice blast and rice leaf spot and the pyrazophos at 750 ppm against blast; showed that the alternation of pyrazophos and T. harzianum reduced blast at a rate similar to that noted when pyrazophos is used alone (that is, respectively 90.5 and 89.1%). This percentage is better than that recorded following treatment by T. harzianum alone (78.4%). Mancozeb alternated with T. harzianum reduced blast at a rate of (83.49%) compared with the fungicide or the antagonist alone (77 and 78.4%). Nevertheless, essential knowledge concerning the distribution and efficacy of Trichoderma species as antagonists for their effective use to act against rice blast pathogens is lacking in Mali. That is why, the present research was undertaken to explore the possibility of isolating and selecting Trichoderma strains with high biocontrol activity against rice blast disease pathogen.
MATERIALS AND METHODS
Source of isolates and pathogen isolation
Samples of contaminated soil (5) from rice fields (lowland), leaves (15) and panicles (3) of diseased rice were collected at the Longorola research station of the ²Institut d’Economie Rurale (IER)² at the ²Centre Regional de Recherche Agronomique (CRRA)² of Sikasso. Rice plant samples were collected from both leaf infections. Besides rice, samples were also obtained soil samples from rice growing fields in Sikasso.
Isolation of pathogen
Blast lesions were surface sterilized with 0.1% mercuric chloride for 1 min, washed with sterilize distilled water and placed over clean glass slides kept in sterile Petri dishes padded with moist cotton. The Petri dishes were incubated for 48 h at room temperature (28 ± 2°C). Single spore method was used for purification (Noman et al., 2018; Zhang et al., 2013). For that, single conidia were identified from the sporulating lesions using a stereomicroscope and aseptically transferred to a petri dish (PDA media). The isolated and purified strain was identified mainly from its macroscopic characters of its colonies and microscopic characters of its mycelium on the basis of identification keys (Botton et al., 1990).
Media suitable for culturing P. oryzae
The determination of suitable media for culturing P. oryzae (M. oryzea) was done according to the method described by Vanaraj et al. (2013). The M. oryzae Longorola strain (M. oryzae LS) was grown on PDA for 10 days at room temperature. From the margin of actively growing fungus, 5 mm discs were plugged out. Sterile Petri dishes containing PDA, oat meal agar, rice agar, rice polish agar and malt extract agar were inoculated each with a single 5 mm disc of the fungus and incubated at room temperature for 30 days. Four replications were maintained for each medium. The fungal growth was measured at 5-day-intervals until 30 days. Further, the colony characters of the single isolate on different media were recorded on 30th day. All the 11 isolates were grown on PDA and their colony morphology was observed.
Spore induction on stem bits
As the fungus grows and sporulates slowly on rice, but sporulate more quickly on maize and P. ripens and we need more spores for the pathogenicity test; we tested the spore production ability of the fungus on these plants. For that, the stem bits from maize, rice (20-day old crop) and P. repens were collected from the field and cut into small pieces of 1 cm in length. 15 pieces were placed in 50 ml Erlenmeyer flasks and sterilized for 1 h and 30 min. Each flask was inoculated with two 5 mm diameter mycelial discs of the Longorola isolate and incubated for 15 days at room temperature (Vanaraj et al., 2013). Three stem pieces were sampled at 5, 10 and 15 days after inoculation (DAI). Each stem piece was placed in a test tube containing 1 ml of sterile water, shaken well to dislodge the spores and decanted. The spore concentration was assessed using a haemocytometer.
Measurement of spore size
After spore induction on bits tests shows that the fungus grows and sporulates more quickly on maize bits than on rice and P. repens. The M. oryzae LS isolate was multiplied on maize stem bits for 15 days and spores were collected by placing stem piece in a test tube containing 1 ml of sterile water, shaking well to dislodge the spores and decanting. The length and width of 10 spores were measured for each isolate using a micrometer as in Vanaraj et al. (2013).
Isolation and identification of Trichoderma sp.
One gram of the soil sample was taken and added to 1 ml of sterilized distilled water to make a dilution of 10-1. This suspension was then subjected to serial dilutions and a dilution of 10-5 was attained. One milliliter of each dilution viz., 10-3 to 10-4 was poured on to Trichoderma Specific Medium (TSM) (Elad et al., 1981) and purified by single spore method (Zhang et al., 2013). The TSM medium is composed of the following constituents (g/L): MgSO4 9; 7 H2O, 0.2; K2HPO4, 0.9; KCl, 0.15; NH4NO3, 1.0; glucose, 3.0; chloramphenicol 0.25; p-dimethylaminobenzenediazo sodium sulfonate 0.3; pentachloronitrobenzene 0.2; rose-bengal 0.15; agar 20 Isolates were identified on the basis of their morphological characters, according to conidiophore, shape of the phialides and emergence of phialophores and phialospores (Soesanto et al., 2011). The purified and identified cultures of Trichoderma strains were maintained on PDA medium and stored at 4°C for further use.
Antagonism characterization
Out of 12 isolates of Trichoderma sp. obtained from different soil samples, only 3 strains showed, in pre-test, an antagonistic activity against the rice blast pathogen isolated. These three isolates identified as T. harzianum were tested for antagonism effect against the isolated rice pathogen: M. oryzae. The antagonism studies were done by using dual culture techniques as developed by Rahman et al. (2009). The mycelial bits of 5 mm diameter of Trichoderma sp. strain and pathogen were placed opposite to each other (4 cm) on Petri plates containing PDA. The plates were run in triplicates with one control set maintained without inoculating the Trichoderma sp. isolates. The plates were incubated at 28 ± 2°C for one week, and the growth of the pathogen tested against the 3 isolates of Trichoderma which showed antagonism activity in the pre-test. The data were recorded regularly on the growth of the pathogen and Trichoderma sp. tested. Percentage of mycelial growth inhibition (MGI) was calculated according to the formula:
MGI% = (dc - dt) ×100/dc
Where, dc= fungal colony diameter in control sets, dt= fungal colony diameter in treatment sets.
Correction of the document in français
Plates were tested in triplicate, with a control set maintained without inoculating Trichoderma sp. The plates were incubated at 27+-1°C for one week, and pathogen growth was tested against the 3 Trichoderma isolates that showed antagonistic activity in the pre-test. Data was recorded regularly on the growth of the pathogen and Trichoderma sp. Tested. The percentage inhibition of mycelial growth was calculated according to the formula:
MGI%=(dc-dt)×100/dc
Where, dc=diameter of fungal colony in control sets, dt=diameter of fungal colony in treatment sets.
The inhibition exerted by the genus Trichoderma was estimated by calculating the antagonism coefficient (Morsy, 2005) according to the following formula:
a=(Ctem-Ctrait)/Ctem
Where a is the antagonism coefficient, Ctem the mean radius of the control colonies (strains of phytopathogenic fungi growing in the absence of antagonist), Ctrait the mean radius of the colonies in the presence of the antagonist.
The evaluation of myco-parasitic activity was carried out in accordance with the method of Dabire et al. (2016). This method consisted in carrying out cocultures in direct confrontation between the pathogen and the antagonist for 21 days. After the 21 days, mycelial fragments of the pathogen in the area of intersection were cultured at 25°C for 5 days to observe the viability of the pathogen.
Sandwiched Petri plates, a setup described in Li et al. (2018), was employed to determine if the Trichoderma isolates tested can produce volatile compounds, and how the volatile compounds affect the growth of M. oryzae LS. After inoculating M. oryzae LS and Trichoderma isolates on PDA plate, M. oryzae LS plate was placed on top of Trichoderma plate, sealed with three layers of Parafilm, and incubated at 25°C. Each plate of M. oryzae LS also was sandwiched with an un-inoculated PDA plate (control treatment). Colony diameter of M. oryzae LS was measured 5 days later. We evaluated the inhibitory effect of M. oryzae LS volatile compounds on Trichoderma in the same way except that 5-day-old (after the inoculation of culture plug) M. oryzae LS culture was used to ensure enough M. oryzae LS biomass. Colony diameter of Trichoderma was measured 36 h later. The duration of volatile compound exposure between the two experiments was different because Trichoderma grew much faster than M. oryzae LS. Each treatment included three biological replicates and was repeated three times.
RESULTS AND DISCUSSION
Virulent strains of M. oryzae
In this study, only one isolate of M. oryzae (Figure 1) was obtained from a sample of rice from the Longorola research station of the CRRA in Sikasso (IER), and named M. oryzae Longorola strain (M. oryzae LS).
Growth of M. oryzae was rapid on PDA followed by malt extract agar (Table 1 and Figure 1). At 5 days after inoculation (DAI), the colony diameter was 3.45 cm. The M. oryzae isolate grew 8.95 cm in diameter in the PDA, contained in 9.0 cm Petri dishes, at 10 DAI. On the same day, its growth on the rice agar was 6.95 cm. At 15 DAI, the colony diameter of M. oryzae was 9.0 cm in both media tested.
The spore density was greatest when M. oryzae was propagated on maize followed by the rice stalk at all observation intervals (Table 1). The production of conidia increased over time for both maize and rice stalks. For maize, it was respectively 395, 1630, 2900 at 5, 10 and 15 DAI. This M. oryzae strain produce very few spores on P. repens, a grass which was found support growth and bigger spores of M. oryzae than rice. As maize stalk shows better grow and more spore production, we chose to multiply M. oryzae on maize stalk to have more spore in a relatively short time for tests.
The length and width of M. oryzae conidia (Figure 1) were measured. Results of spore dimension measurement, showed that the mean spore length of the M. oryzae isolate was 21.5 µm, while the mean spore width was 9.6 µm (Table 2).
Virulence of Magnaporthe oryzae LS strain on rice
The strain of M. oryzae LS inoculated on the rice leaves, kept under humidity, showed lesions similar to those of
blast (Figure 2A). The uninoculated control, meanwhile, showed no signs of pathology (Figure 2B).From the lesions of the inoculated rice leaves, we were able to re-isolate the M. oryzae LS strain. These results confirm the pathogenicity of the isolated M. oryzae LS strain.
Strains of Trichoderma sp. isolated and identified
Isolation of M. oryzae antagonist fungi from soil and rice plant samples yielded 12 colonies (Figure 3). These isolates gave colonies typical of Trichoderma sp. on Trichoderma selective medium. These isolates showed very rapid growth, good sporulation, and yellowish-green and green pigmentations on PDA medium. Three isolates from soil 3 (S31, S32 and S33) (Colonies number 1, 2 and 3 in Figure 3) showed very rapid growth and formed 2 concentric rings with green conidial production. The conidia production was denser in center then towards the margins on PDA medium. The mycelia of these 3 isolates are septated. For these 3 isolates, the conidiophore is sparsely branched and carries philaids (Figure 4). In turn, phialides carry sub-globular-shaped spores or conidia. On the basis of their cultural and morphological characters of these three Trichoderma isolates, were identified as T. harzianum (Gams and Bisset, 1998; Shah et al., 2012. These 3 strains, that showed, in a pre-test, an antagonistic activity against M. oryzae LS) are under molecular characterization to confirm the identity of each.
Antagonism tests
The average growth ranges of M. oryzae LS placed in cocultures in direct confrontation with the isolates of T. harzianum are given in Table 3. Analysis of data in Table 3 indicates that all of the T. harzianum isolates caused a significant reduction in the average growth radius of the M. oryzae LS strain tested. The highest coefficient of antagonism obtained was 0.78 obtained with the strain T. harzianum S33 (Table 3), followed by the strain T. harzianum S32 with a coefficient of antagonism of 0.71. The T. harzianum S31 strain gave the lowest coefficient of antagonism estimated at 0.55. The effect of T. harzianum S33 on the mycelium growth of M. oryzae LS is presented in Figure 5. The results on mycoparasitism presented showed that only the T. harzianum S33 demonstrates a clear mycoparasitic activity against M. oryzae LS (Table 4). None of the three Trichoderma strains tested were able to control the growth of the M. oryzae LS strain by producing volatile compounds.
DISCUSSION
Twelve different colonies of Trichoderma sp. were isolated from soil samples from the Longorola station and Sikasso rice fields. Küçük and Vanç (2003) isolated 19 strains of Trichoderma from 31 soil samples, while we isolated 12 from 5 sample from agricultural soil of Sikasso (Mali). Which is an indicator of the richness of these soils compared to those of the Turky? Growing the fungus on pieces of the host's stem is the easiest way to induce sporulation in order to study spores (Vanaraj et al., 2013). M. oryzae spores isolated from rice in this study were significantly smaller than those isolated from the rice fields of Baguineda. Gupta et al. (2020) states that the spore size of the fungus M. oryzae varies among isolates depending on environmental conditions. Measurement of the spore size of different isolates of M. oryzae from rice grown on PDA by Gayatonde et al. (2016) showed that the mean length ranged from 21.2 to 28.4 μm. In our study, the spore length of M. oryzae isolates grown on pieces of corn stalk was 21.5. Vanaraj et al. (2013) observed variations in the length and width of M. oryzae spores due to the effect of artificial media. They also noticed that temperature had no effect on the width of the spore while the length was affected.
The results obtained at the end of this study reveal that out of all the Trichoderma isolated in Mali, only 3 have an inhibitory activity on the mycelial growth of M. oryzae, but to different degrees. Naravanasamv et al. (2015) demonstrated the inhibitory activity of Trichoderma species on the growth of P. oryzae. In this study, in addition to antifungal activity, only the T. harzianum S33 strain exhibited myco-parasitic activity on the M. oryzae isolate tested. In addition, an antagonism coefficient of 0.71 was obtained with this strain. These results are also in the same direction as those of Li et al. (2018) who showed that 5 isolates of T. harzianum tested have a strong inhibitory power against 3 pathogenic fungi, including Fusarium oxysporum f.sp. cepae (82.77%). However, not all of the 5 isolates exerted a myco-parasitic action on all the pathogens tested and the intensity of the inhibitions varied from one pathogen to another and from one antagonist to another but values antagonism coefficients greater than 0.75 were obtained with two of the isolates tested. The antagonism in the distance confrontation was much lower than in the direct confrontation. This could be explained by the fact that T. harzianum uses several modes of action and depending on the nature of the pathogen to exert their antagonistic power (Benitez et al., 2004; Sood et al., 2020). According to Benhamou and Chet (1997) fungi chitin is an essential constituent of the wall which surrounds and protects cells from the environment. In the same way, Latgé (2007) showed that the cell wall is essential for fungal growth and for the resistance of the fungus to external attacks. Its alteration linked to the action of Trichoderma would lead to an alteration of the mycelium which results in aggregation, retraction and vacuolation of the cytoplasm. The statements by Benhamou and Chet (1996) and Latgé (2007) were confirmed by Tapwal et al. (2015), who indicate that the inhibitory power of Trichoderma species is manifested by a significant lysis of the mycelial cells of the pathogens. Nusaibah and Musa (2019), reports that Trichoderma species have the ability to attack pathogens via different modes of action. They can use the antibiotic which results from the production of substances which act as "antibiotics" and which inhibit the growth of the pathogen. In some cases, the colony of T. harzianum grows on that of the pathogen. According to Sharma (2011), these types of interactions indicate competition and parasitism, respectively. The results obtained in remote confrontation mode indicate that the use of volatile compounds was not the main path that explains the observed antagonism.
CONCLUSION
Only one pathogenic isolate of M. oryzae was isolated from soil and diseased rice plant samples from Sikasso, Mali. Twelve isolates of Trichoderma were isolated from soils samples. Out of these isolates: T. harzianum S31, T. harzianum S32 and T. harzianum S33 showed high antagonism activity against M. oryzae LS isolated from Mali.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGEMENTS
The authors appreciate the ²Fond Compétitif pour la Recherche et l’Innovation Technologique (FCRIT)² for the financial support of this study, and the ²Centre National de Recherche Scientifique et Technologique (CNRST)² for the fund management and their support.
REFERENCES
Alfiky A, Weisskopf L (2021). Deciphering Trichoderma-plant-pathogen interactions for better development of biocontrol applications. Journal of Fungi 7(61):1-18. |
|
Asibi AE, Chai Q, Coulter JA (2019). Rice Blast: A Disease with Implications for Global Food Security. Journals of Agronomy 9(8):1-14. |
|
Bastakoti S, Belbase S, Manandhar S, Arjyal C (2017). Trichoderma species as Biocontrol Agent against Soil Borne Fungal Pathogens. Nepal Journal of Biotechnology 5(1):39-45. |
|
Benhamou N, Chet I (1996). Parasitism of sclerotia od sclerotium Rolfsii by Trichoderma harzianum: ultrastuctural and cytochemical aspects of the interaction. Phytopath 86(4):405-416. |
|
Benhamou N, Chet I (1997). Cellular and molecular mechanisms involved in the interaction between Trichoderma harzianum and Pythium ultimum. Applied and Environmental Microbiology 63(5):2095-2099. doi: 10.1128/aem.63.5.2095-2099.1997. |
|
Benitez T, Ana M, Rincón M, Carmen LA, Codón C (2004). Biocontrol mechanisms of Trichoderma strains. International Microbiology 7(4):249-260. |
|
Botton BBA, Fevre M, Gauthir S, Guy PH, Larpent JP, Reymond P, Sanglier JJ, Vayssier Y, Veau P (1990). Beneficial and detrimental harvests of industrial importance. 2éme édition. masson collection biotechnologies pp. 34-42. |
|
Chou C, Nancy CN, Hadi B, Tanaka T, Chiba S, Sato I (2019). Rice blast management in Cambodian rice fields using Trichoderma harzianum and a resistant variety. Crop Protection 135:104864. |
|
Chuaki T, Lavarde V, Lachaud L, Henneken C (2003). Invasive Infections Due to Trichoderma Species: Report of 2 Cases, Findings of In Vitro Susceptibility Testing, and Review of the Literature. Clinical Infectious Diseases 35(11):1360-1367. |
|
Dabire TG, Bonzi S, Somda I, Legreve A (2016). Evaluation of the Potential of Trichoderma harzianum as a Plant Growth Promoter and Biocontrol Agent Against Fusarium Damping-off in Onion in Burkina Faso. Asian Journal of Plant Pathology 10(4):49-60. |
|
Dougoud J, Clottey V, Bateman M, Wood A (2018). Study on crop protection in countries where the Green Innovation Centres for the Agri-Food Sector programme is active. Country report for the 'Green Innovation Centre' (GIC) in Mali P 113. |
|
Elad Y, Chet I, Henis YA (1981). A selective medium for improving quantitative isolation of Trichoderma spp. from soil. Phytoparasitica 9(1):59-67. |
|
Es-Soufi R, Tahiri, H, Azaroual L, El Oualkadi A, Martin P, Badoc A, Lamarti A (2020). Biocontrol Potential of Bacillus amyloliquefaciens Bc2 and Trichoderma harzianum TR against Strawberry Anthracnose under Laboratory and Field Conditions. Agricultural Sciences 11(3):260-277. |
|
Food and Agriculture Organization Corporate Statistical Database (FAOSTAT) (2019). Statistical Database. Available online: |
|
Gams W, Bissett J (1998). Morphology and Identification of Trichoderma. In: Trichoderma and Gliocladium: Basic Biology, Taxonomy and Genetics, Harman GE, Kubicek CP. Taylor and Francis, London, UK. ISBN-13: 9780203483558 1:3-34. |
|
Gayatonde V, Mahadevu P, Vennela PJ (2016). Study of suitable culture media and other abiotic factors for the growth and sporulation of Magnaporthe Oryzae. Ecology, Environment and Conservation 22(2):297-301. |
|
Gupta DR, Surovy MZ, Mahmud NU, Chakraborty M, Paul SK, Hossain MS, Bhattacharjee P, Mehebub MS, Rani K, Yeasmin R, Rahman M, Islam MT (2020). Suitable methods for isolation, culture, storage and identification of wheat blast fungus Magnaporthe oryzae Triticum pathotype. Phytopathology Research 2(1):1-13. |
|
Gurr S, Samalova M, Fisher M (2011). The rise and rise of emerging infectious 643 fungi challenges food security and ecosystem health. Fungal Biology Reviews 25(4):181-188. |
|
Khemmuk W (2016). Plant pathogenic Magnaporthales in Australia, with particular reference to Pyricularia oryzae on wild and cultivated rice. Master thesis presented at the University of Queensland, Austria 193 p. |
|
Küçük C, Vanç M (2003). Isolation of Trichoderma Spp. and Determination of Their Antifungal, Biochemical and Physiological Features. Turkish Journal of Biology 27:247-253. |
|
Latgé JP (2007). The cell wall: A carbohydrate armour for the fungal cell. Molecular Microbiology 66(2):279-290. |
|
Li N, Alfiky A, Wang W, Islam M, Nourollahi K, Liu X, Kang S (2018). Volatile Compound-Mediated Recognition and Inhibition between Trichoderma Biocontrol Agents and Fusarium oxysporum. Frontiers in Microbiology 9(2614):1-16. |
|
Morsy EM (2005). Role of growth promoting substances producing microorganisms on tomato plant and control of some root rot fungi. Ph.D. Thesis, Fac. Agric. Ain shams Univ., Cairo, Egypt. |
|
Mouria A, Mouria B, Hmouni A, Touhami, AO, Douira A (2018). Integrated Control against Rice Blast and Leaf Spot by Trichoderma harzianum and Two Fungicides. Annual Research and Review in Biology 29(1):1-6. |
|
Naravanasamv P, Thokala P, Muthkrishnan S, Kamil D (2015). Screening of different Trichoderma species against agriculturally important foliar plant pathogens. Journal of Environmental Biology 36(1):191-198. |
|
Noman E, Al-Gheethi AA, Rahman NA, Talip B, Mohamed R, Nagao H, Kadir OA (2018). Single spore isolation as simple and efficient technique to obtain fungal pure culture, Conference series Earth Environmental Science 140(1):012155. |
|
Nusaibah SA, Musa H (2019). A Review Report on the Mechanism of Trichoderma spp. as Biological Control Agent of the Basal Stem Rot (BSR) Disease of Elaeis guineensis, Trichoderma - The Most Widely Used Fungicide, Mohammad Manjur Shah, Umar Sharif and Tijjani Rufai Buhari, IntechOpen. |
|
Pordel A, Ravel S, Charriat F, Gladieux P, Cros-Arteil S, Milazzo J, Henri AH, Javan-Nikkhah M, Mirzadi-Gohari A, Moumeni A, Tharreau D (2021). Tracing the Origin and Evolutionary History of Pyricularia oryzae Infecting Maize and Barnyard Grass. Phytopathology 111(1):128-136. |
|
Rahman MA, Begum MF, Alam MF (2009). Screening of Trichoderma Isolates as a Biological Control Agent against Ceratocystis paradoxa Causing Pineapple Disease of Sugarcane. Mycobiology 37(4): 277-285. |
|
Rivera-Méndez W, Obregón M, Morán-Diez ME, Hermosa R, Monte E (2020). Trichoderma asperellum Biocontrol Activity and Induction of Systemic Defenses against Sclerotium cepivorum in Onion Plants under Tropical Climate Conditions. Biological Control 141:104145. |
|
Savary S, Willocquet L, Elazegui FA, Castilla NP, Teng PS (2000). Rice 706 pest constraints in tropical Asia: Quantification of yield losses due to rice pests in a range of 707 production situations. Plant Disease 84(3):357-369. |
|
Shah S, Nasreen S, Sheikh PA (2012). Cultural and Morphological Characterization of Trichoderma spp. Associated with Green Mold Disease of Pleurotus spp. in Kashmir. Research Journal of Microbiology 7(2):139-144. |
|
Sharma P (2011). Complexity of Trichoderma-Fusarium interaction and manifestation of biological control. Australian Journal of Crop Science 5(8):1027-1038. |
|
Soesanto L, Utami DS, Rahayuniati RF (2011). Morphological characteristics of four Trichoderma isolates and two endophytic Fusarium isolates. Canadian Journal on Scientific and Industrial Research 2(8). |
|
Sood M, Kapoor K, Kumar V, Sheteiwy MS, Ramakrishnan M, Landi M, Araniti F, Sharma A (2020). Trichoderma: The "Secrets" of a Multitalented Biocontrol Agent. Plants 9(6):762-787. |
|
Soullier G, Demont M, Arouna A, Lançon F, Mendez del Villar P (2020). The state of rice value chain upgrading in West Africa. Global Food Security 25:100365. |
|
Swain H, Adak T, Mukherje AK, Mukherjee PK, Bhattacharyya P, Behera S, Bagchi TB, Patro R, Khandual MK, Bag TK Dangar S, Jen LJ (2018). Novel Trichoderma strains. isolated from tree barks as potential biocontrol agents and biofertilizers for direct seeded rice. Microbiological Research 214:83-90. |
|
Tapwal A, Tyagi A, Thakur G, Ghandra S (2015). In-vitro evaluation of Trichoderma species against seed borne pathogens. International Journal of Biological and Chemical Sciences 1(10):14-19. |
|
Tembo B, Mulenga RM, Sichilima S, Kenneth K, Moses M, Patrick CC, Pawan KS, Xinyao H, Kerry FP, Gary LP, Ravi PS, Hans JB (2020). Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia. PLoS ONE 15(9):e0238724. |
|
U.S. Department of Agriculture (USDA) (2012). National Agricultural Statistics Service, "Crop Produc¬tion," |
|
Vanaraj P, Kandasamy S, Ambalavanan S, Ramalingam R, Sabariyappan R (2013) Variability in Pyricularia oryzae from different rice growing regions of Tamil Nadu, India. African Journal of Microbiology Research 7(26):3379-3388. |
|
Woo SL, Ruocco M, Vinale F, Nigro M, Marra R, Lombardi N, Pascale A, Lanzuise S, Manganiello G, Lorito M (2014). Trichoderma-based Products and their Widespread Use in Agriculture. The Open Mycology Journal 8(Suppl-1, M4):71-126. |
|
ZEF, FARA, IER (2017). Country Dossier: Innovation for Sustainable Agricultural Growth in Mali. Program of accompanying research for agriculture innovation. Bonn and Accra. Centre for research, Forum for agricultural research in Africa and Council for scientific and industrial research. |
|
Zhang K, Su Y-Y, Cai L (2013). An optimized protocol of single spore isolation for fungi. Mycologie 34(4):349-356. |
Copyright © 2025 Author(s) retain the copyright of this article.
This article is published under the terms of the Creative Commons Attribution License 4.0