Bioactivity of Trichoderma harzianum isolates against the fungal root rot pathogens with special reference to Macrophomina phaseolina causing dry root rot of mungbean

In the rice-fallow system, dry root rot (DRR) is an emerging disease of mungbean (Vigna radiata (L.) R. Wilczek var. radiata) caused by the necrotrophic fungus Macrophomina phaseolina. The pathogen causes extensive production losses. In this study, the bioactivity of four Trichoderma harzianum isolates, namely Th-Dharwad, Th-Raichur, Th-Niphm, and Th-Udaipur procured from the Indian research institutes were evaluated against M. phaseolina of mungbean by the dual culture technique. The efficacy of these T. harzianum isolates were also compared with the effective fungicides such as thiram and carbendazim by the poison food method. Results showed that among the T. harzianum isolates, isolate of Th-Raichur was most effective, exhibiting 76.96% mycelial growth inhibition of the test pathogen. As compared to the thiram, carbendazim was more effective, and exhibited 100% mycelial growth inhibition of the test pathogen. In addition, carbendazim was also more effective than the isolate of Th-Raichur. In the sick pot experiment, mungbean seeds treated with Th-Raichur isolate showed a lower percent incidence of DRR (20%) than the untreated seeds (86.6%). The biological spectrum of Th-Raichur isolate was examined against M. phaseolina isolated from the different hosts such as urdbean and vegetable soybean, alongwith two other root pathogens, namely Fusarium solani of mungbean, and Sclerotium rolfsii of urdbean. The isolate of Th-Raichur showed maximum antagonistic activity against the pathogens M. phaseolina and F. solani of mungbean. Thus, Th-Raichur isolate can be used as a potential fungal biocontrol agent for the reduction of DRR in mungbean.

The fungus Macrophomina phaseolina (Tassi) Goid is most prevalent among the crop species, including mungbean (Vigna radiata (L.) R. Wilczek var. radiata), urdbean (Vigna mungo (L.) Hepper) and vegetable soybean (Glycine max (L.) Merr.), and is accountable for the significant losses in the production (Iqbal and Mukhtar 2014). In addition to M. phaseolina, other root rots pathogens, such as Fusarium solani (Mart.) Sacc. and Sclerotium rolfsii Sacc (syn. Athelia rolfsii (Curzi.) Tu & Kimbr) are also cause 30-40% of losses to the pulses, particularly in mungbean and urdbean (Biswas and Sen 2000;Rojo et al. 2007). Mungbean is an important pulse crop, grown as a rotation crop in the cereal farming system in Asia, particularly in the rice-fallows. Dry root rot (DRR), sometimes named as Charcoal rot for soybean and sesame (Cardona and Rodriguez 2006), incited by M. phaseolina (asexual stage of Rhizoctonia bataticola), is widespread, and an important emerging disease of mungbean in South Asia, causes 27-44% production loss of mungbean during the favourable conditions (Iqbal and Mukhtar 2014).
The pathogen is a soil and seed-borne, infects entire parts of the mungbean plants at all growth stages, but it is generally most destructive and active in the fibro vascular systems of roots (Khan 2007). The sclerotial characteristics of this pathogen make the management of this disease difficult for mungbean growers (Kumari et al. 2012). For the mitigation of DRR in mungbean, several methods, such as 1 3 seed treatment with chemical fungicides (like carbendazim) and bio-fungicides, use of the resistant varieties Choudhary et al. 2011;Pandey et al. 2020), and soil amendment by microbial biocontrol agents/fungicides have been adopted. Although, chemical fungicides showed potential results in the mitigation of M. phaseolina, but excess fungicide residues creates the environment and human health risks (Thilagavathi et al. 2007). To overcome fungicide risk problems, biological control is one of the sustainable and eco-friendly methods for suppressing the plant diseases.
The use of fungal antagonists, such as Trichoderma harzianum Rifai, T. viride Pers., and T. hamatum (Bonord.) Bainier in the soil, and as a seed dressing, has shown potential control of DRR of mungbean (Kumari et al. 2012;Deshmukh et al. 2016), and other root rot pathogens, namely Rhizoctonia solani and Fusarium solani (Khan et al. 2014;. However, little information is available on the multipathogenic activity of Trichoderma species. In the view of reported antagonism of M. phaseolina by Trichoderma species, in the present study, screening of four isolates of T. harzianum procured from different Indian research institutes were investigated in the laboratory. In addition, biological spectrum of the most effective T. harzianum isolate was studied against the other root rot pathogens of mungbean, urdbean and vegetable soybean in order to find out the fungal biocontrol agent having multi-pathogenic activity. Efficacy of the most effective T. harzianum isolate in the management of DRR has also been carried out by the sick pot method in a glasshouse. In the present study, four isolates of T. harzianum were procured from the Department of Plant Pathology of Indian Research Institutions, namely University of Agricultural Sciences Dharwad, Karnataka, National Institute of Plant Health Management, Rajendranagar, Hyderabad, University of Agricultural Sciences Raichur, Karnataka, and Maharana Pratap University of Agriculture and Technology, Udaipur City, Rajasthan state. These isolates were named as Th-Dharwad, Th-Niphm, Th-Raichur, and Th-Udaipur, respectively. These isolates were further verified by studying their cultural characteristics on the PDA (Potato dextrose agar) medium (200 g: Potato infusion, 20 g: dextrose, 15 g: agar, 1 L: distilled water), and through the morphological characteristics with the help of a microscope (stereo-binocular, Nikon-ci-digital, Olympus) by studying the morphology of conidia; conidiophores types, and with the aid of fungal keys. Cultural characteristics like colour of the colony, and its appearance and growth rate of each isolate of T. harzianum were examined following the procedures of Gams and Bisset (1998). Procured cultures were sub-cultured on PDA slants, and were preserved at 4 °C for further need.
DRR symptomatic mungbean plants were uprooted from the mungbean field of World Vegetable Center South Asia, Hyderabad, India (N 17°′, E 078°, Elevation: 550 m) during Kharif (rainy season) 2017 and brought to the laboratory in pre-sterilized polyethylene bags. Symptomatic roots of the collected plants' samples were cut into minute pieces and sterilized (surface) with Clorox solution (2%) for 2 min. The small pieces of disease tissue (sterilized) were placed on the surface of pre-poured PDA medium in Petri plates in three replicates. The PDA plates inoculated with disease samples were incubated for 6-7 days at 28 °C. The fungal colonies appeared after the incubation periods were purified on separate agar plates. Identification of the pathogen was conducted following the standard mycological procedures (Dhingra and Sinclair 1978). Cultural and morphological characteristics of M. phaseolina were carried out. The pathogen was identified based on its mycelium structure and size of the microsclerotia, and with the help of fungal key. Likewise, M. phaseolina from urdbean and vegetable soybean roots, F. solani from mungbean root, and S. rolfsii from urdbean root were isolated and identified by using standard mycological procedures, separately. The isolates were named as MPM for M. phaseolina of mungbean, MPU for urdbean, MPS for vegetable soybean, FSM for F. solani of mungbean, and SRU for S. rolfsii of urdbean. The culture of each pathogen was maintained on the slants at 4 °C after purification by single spore/sclerotial method for further investigation, separately. Isolates of M. phaseolina isolated from vegetable soybean, mungbean, and urdbean were also identified through the molecular method of sequencing of the ITS region (ITS1, 5.8S and ITS2) of the nuclear rDNA operon (Pandey et al. 2020).
Two fungicides viz., thiram 75% WP and carbendazim 50% WP used in this study were purchased from the local vendor of Hyderabad, India. Both fungicides (5 mg/ml) were directly mixed with the PDA medium before pouring into the Petri plates. Each Petri plate contained 10 ml medium.
The antagonistic activity of T. harzianum isolates against M. phaseolina of mungbean was assessed in the laboratory through the dual culture technique (Dennis and Webster 1971), while fungicides were evaluated through the poison food method (Grover and Moore 1962). In dual culture technique, mycelial disc (6 mm) of 7-day-old culture of the test pathogen was placed on a Petri plate (80 mm diam.) one cm away from the edge containing 10 ml pre-poured solidified PDA medium. At the opposite side of the Petri plate, a disc of T. harzianum (6 mm) of each isolate was placed separately. In the poison food method, 5 mg/ml of each fungicide was added to the PDA medium (10 ml) in pre-sterilized Petri plates, separately. These plates (poisoned) were inoculated with the test pathogen (6 mm diam.) cut from periphery of the 7-day-old culture. In control plate, the mycelial disc of test pathogen was inoculated on the agar surface which did not contain any treatment. The control and inoculated plates were arranged in a complete randomized design (CRD) in three replicates, and were kept in a BOD incubator (28 °C).
After 7 days of incubation, percent mycelial growth inhibition of the test pathogen by T. harzianum isolates and fungicides was recorded by the formula PGI = C − T/C × 100; where PGI is the percent growth inhibition, C is the mycelial diameter of the test pathogen in control plates (mm), T is the mycelial diameter of test pathogen in treatment plates -antagonist/fungicide (mm).
During the sick pot experiment, sorghum grains were used for multiplication of M. phaseolina isolated from mungbean (Choudhary et al. 2011). The fungal colonized sorghum grains were made in powder form by grinding it in a blender and 50 g of prepared powder (the inoculum) was mixed with 1 kg of sterilized soil (autoclaved black sandy). For the proper fungal colonization, the inoculated soil mixture was incubated at room temperature for one week. After one week of the incubation, Macrophomina colonized soil mixture (2 kg) was placed into plastic pots. Ten seeds of DRR susceptible mungbean line (VC3960-88) were sown in a pot and the pot was kept in a glasshouse (33 ± 2 °C). In total, 9 sick pots were prepared. The soil moisture was maintained in the 60% water holding capacity. Once the mortality of mungbean plants was reached > 80%, these sick pots were used for the disease screening. Ten seeds of the susceptible mungbean line (VC3960-88) were treated with most effective T. harzianum isolate @ 4 g/kg and carbendazim @ 2 g/ kg by seed dressing method, separately. The treated seeds were sown in the individual pots. In control pots, the sterilized seeds were sown in sick soil. Treated and control pots were set in a randomized complete block design (RCBD) in triplicates, and were kept in a glasshouse. When the mortality of plants in control pots were reached above 80%, then the percent disease incidence (PDI is the number of wilted plants due to DRR/Total number of healthy plants × 100) was recorded at maturity in all the treated, and control pots.
The biological spectrum of the most effective isolate of T. harzianum was carried out against other root rot pathogens of vegetable legumes, namely M. phaseolina from urdbean (MPU) and vegetable soybean (MPS), F. solani from mungbean (FSM), and S. rolfsii from urdbean (SRU) including M. phaseolina from mungbean (MPM). Antagonistic potency of the effective T. harzianum isolate against these pathogens was assessed by the dual culture technique as described earlier. The control and inoculated plates of each test pathogen were kept separately in a BOD incubator in three replicates in CRD at 28 ºC. After 7 days of incubation, the percent mycelial inhibition of each fungal pathogen by the fungal antagonist was recorded.
In the present investigation, all the experiments were carried out in three replications and a completely randomized design (CRD) was used for the in vitro experiment, and randomized complete block design (RCBD) for the glasshouse experiment. The percent observations were angularly transformed prior to the statistical analysis. The 9.4 version of SAS software was used for the analysis of variance (ANOVA) of the data.
On the PDA medium, procured isolates of T. harzianum produced green colonies in 1-2 concentric rings. Dense conidia were produced in the center of the plates, and light towards the margins. The isolate from NIPHM had a slower growth rate. After 5 days, the mycelial growth diameters were 25 × 30, 35 × 35, 35 × 35, and 35 × 35 mm for Th-Niphm, Th-Udaipur, Th-Raichur, and Th-Dharwad, respectively. The conidia of T. harzianum (average diam. 2.8 × 2.6 µm) were globose to sub globose in 7 days old culture, and were light green in colour.
On the PDA medium, M. phaseolina colonies were initially whitish and later become dark brown to greyish in colour. On the culture plate, Macrophomina produced abundant aerial mycelium with sclerotia that were within the hyphae or immersed on the agar. The mycelium of the fungus was hyline, septate; microsclerotia black, globose and were 100-120 µm in size. Macrophomina phaseolina isolate of urdbean had feathery mycelial growth, while Macrophomina from vegetable soybean and mungbean had a dense growth on the agar plates. The growth of S. rolfsii was silky-whitish, fast growing on the agar plate with small dark brown mustered like sclerotia (0.4-1.9 mm diam.). Microscopically, they consisted of ribbon-like hyphae. On the PDA plate, colonies of F. solani were creamy whitish, and microscopically, macroconidia were slightly curved with 3-5 septa. The microconidia were oval to kidney shaped (Cho et al. 2001).
The procured four isolates of T. harzianum showed significant antagonistic potency against M. phaseolina causing mungbean DRR. Among the four T. harzianum isolates, isolate from Raichur showed highest antagonistic activity by exhibiting 76.96% mycelial growth inhibition followed by isolates Th-Dharwad, Th-Niphm, while the isolate of Th-Udaipur (66.67%) showed least antagonistic activity in the dual culture assay (Table 1). Among the fungicides, carbendazim was most effective and showed 100% mycelial growth inhibition than that of thiram (69%). In addition, carbendazim was also found to be more effective than the tested T. harzianum isolates ( Table 1). The ANOVA analysis showed a significant (p < 0.0001) variation in the antagonistic activity of T. harzianum isolates. The statistical analysis of data showed that the efficacy of Th-Raichur was significantly different from Th-Udaipur and fungicide carbendazim (Table 1), however not significantly different from the Th-Niphm and Th-Dharwad isolates at the 5% level of significance.
In the sick pot experiment, lower percent DRR incidence was reported for carbendazim (13.3%), and Th-Raichur (20%) treatments, and these results were significantly (p < 0.0001) different from the control set, where 86.6% disease incidence was reported due to DRR (Fig. 1a). The phenotypic DRR disease reaction of the control and treatment (Th-Raichur) sets is shown in Fig. 1b, which reveals that control pots had a higher number of wilted mungbean plants, than the treated pots.
The biological spectrum of the Th-Raichur isolate was studied against M. phaseolina of mungbean (MPM), urdbean (MPU) and vegetable soybean (MPS) inciting DRR, S. rolfsii of urdbean (SRU) causing collar rot, and F. solani of mungbean (FSM) causing root wilt. The isolate of Th-Raichur showed a variable antagonistic activity against the root pathogens. The Th-Raichur isolate was most effective against M. phaseolina of mungbean exhibited 76.44% mycelial growth inhibition followed by F. solani (66.92%), while least effective against Macrophomina of vegetable soybean (50.12%). Additionally, 55-56% mycelial growth inhibition was reported against M. phaseolina and S. rolfsii of urdbean ( Table 2). The statistical analysis of data showed that, the efficacy of the Th-Raichur isolate was significantly (p < 0.0001) different against tested root rot pathogens (Table 2), except for the F. solani of mungbean at the 5% level of significance.
The root rot pathogens in vegetable legumes are of serious concern, causing great loss in the production. The management of these pathogens is very difficult, as their sources of inoculum are either from seeds or from the soil. Therefore, soil amendment or seed dressing by fungicides/ microbial biocontrol agents are the best options to cope with these types of root rot pathogens. Control of these pathogens through the use of fungal biocontrol agents has been regarded as an eco-friendly satisfactory natural substitute to the present chemical fungicides (Bubici et al. 2019). Additionally, due to the eco-friendly nature of biocontrol agents, they are compatible with host plant resistance methods in the integrated disease management program. There are several microbial biocontrol agents have been reported effective, among them, Trichoderma species performed potential antagonistic activity against the plant pathogens, because of its ability to reduce the diseases in legume crops (Vinale et al. 2008a;Dubey et al. 2011). In the present study, we have evaluated the antagonistic activity of four isolates of T. harzianum against M. phaseolina inciting DRR of mungbean. In addition, T. harzianum isolate that showed the more efficiency against Macrophomina was screened against the other root rot pathogens, such as M. phaseolina from urdbean and vegetable soybean, F. solani from mungbean and S. rolfsii from urdbean. Trichoderma harzianum showed more potent activity against M. phaseolina and F. solani of mungbean than that of other root rot pathogens.
Earlier reports showed that T. harzianum efficiently controlled DRR in pigeon pea (Lokesha and Benagi 2007), chickpea (Khan et al. 2014) and other legume crops (Khan and Anwer 2011). Notably, Sreedevi et al. (2011) reported that T. harzianum and T. viride reduced respective 64.4 and 61.1% growth of M. phaseolina (inciting root rot of groundnut) through the dual culture method. Similarly, in the laboratory experiment T7 and T14 isolates of T. harzianum inhibited the growth of M. phaseolina causing charcoal rot in soybean (Khaledi and Taheri 2016). In addition, T. harzianum has also been inhibited the growth of S. rolfsii (92%) causing root rot in groundnut (Biswas and Sen 2000). However, no report is available on the activity of T. harzianum against S. rolfsii from urdbean. In this study, the efficacy of Th-Raichur isolate against S. rolfsii reported, was less than the efficacy of T. harzianum reported by Biswas and Sen (2000). The variation in efficacy of T. harzianum may be due to the different strains of pathogens from the different host species or, different origin of antagonists used during the study.
Recently, Khan et al. (2019) reported that the root rot disease complex of mungbean caused by R. solani and M. phaseolina was controlled by T. harzianum, where only 5-15% disease incidence was reported. In the present study, Th-Raichur isolate also controlled DRR of mungbean, exhibited lower disease incidence than that of the control sets. Trichoderma harzianum isolate also significantly controlled peanut brown root rot caused by F. solani with boosted yield, and was more effective than the T. longibrachiatum isolate (Rojo et al. 2007). Similarly, in the present study, Th-Raichur isolate also significantly inhibited the growth of F. solani of mungbean, has not been reported in the earlier study. Thus, we can see that T. harzianum has a wide range of potency to cope against M. phaseolina, and other root rot pathogens. In particular, the potential activity of T. harzianum may be due to the presence of novel secondary metabolite compounds, such as hydrazinopyridine, gliotoxin, viridin, β-glucanase, harziandione, β-glucosidase, peptaibols, and trichodermin present in Trichoderma species that perform an important  (Harman 2006;Vinale et al. 2008b). The antagonistic activity of Trichoderma may be also due to the presence of cell wall degrading enzymes, such as chitinase, β-1, 3 glucanase, cellulose and protease, which have a major role in the mycoparasitism (Gajera et al. 2012). Furthermore, Trichoderma species also induce systemic acquired resistance (Khaledi and Taheri 2016).
In the earlier investigation, carbendazim has been reported as a potent fungicide against the several root rot pathogens (Shahid and Khan 2016). Similar results were reported in the present investigation, carbendazim was more effective than the thiram, absolutely inhibited the mycelial growth of M. phaseolina, and also was more effective than the four isolates of T. harzianum. Khan et al. (2019) investigated that carbendazim and T. harzianum were equally efficacious against M. phaseolina. On the contrary, in the present study, carbendazim was more effective than the Th-Raichur isolate in terms of both mycelial growth inhibition, and in controlling DRR. This may be due to the virulent nature of T. harzianum isolate used from the different origins.
Thus, from the above findings, we can conclude that Th-Raichur isolate can be used as an alternative of carbendazim to manage DRR of mungbean. Although, Th-Raichur isolate was effective against root rot pathogens of mungbean, but it lacked the diverse resistance to the various root rot pathogens of urdbean, and vegetable soybean. Therefore, further studies are required to find out T. harzianum isolates that have a broad range of antagonistic activity against the root rot pathogens. Additionally, at the different locations, field experiments are required before the recommendation of conventional exploitation of Th-Raichur isolate for managing root rot pathogens of mungbean.  Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.