Combined Application of Trichoderma longibrachiatum T(SP)-20 and Trichoderma asperellum T(AR)-10 in the Management of Stem Rot of Groundnut

Groundnut (Arachis hypogaea L.) is known as king of oil seeds and it is cultivated mainly in China, Indian, Nigeria, Sudan and Myanmar countries. In India, it occupies an area of 4.73 million ha with a production of 6.72 million tonnes (FAOSTAT, 2019). Gujarat ranks first in groundnut area (20 lakh ha) and production (26 lakh tonnes). The highest productivity of groundnut (1604 kg/ha) is recorded in the State of Tamil Nadu, while in Gujarat, productivity is about 1190 kg/ha. Groundnut is suffered by various fungal, bacterial and viral diseases. Among them, stem rot disease was caused by a necrotrophic, soil-borne fungal pathogen, Sclerotium rolfsii Sacc. (Athelia rolfsii), recording a range of yield losses of 10-40% (Dodia et al., 2019). The symptoms are partial or complete wilting of the stem and branches. As the pathogen produces a very hard resting structure sclerotia having melanin pigment on the outer membrane and a long-lasting survival period it is too difficult to manage this disease by applying a single strategy. Due to undefined usage of systemic fungicides most of the pathogens develop resistance in addition to this, fungicides leave some toxic residual compounds in plant parts. Biocontrol is an alternative method under which Trichoderma spp. play a crucial role in the management of soil-borne diseases. Trichoderma spp. exhibit different mechanisms like competition, antibiosis, mycoparasitism, lysis and induced systemic resistance. Lytic enzymes such as chitinase and glucanase produced by the Trichoderma spp. effectively inhibited the Sclerotium rolfsii (S. rolfsii) (Kumar et al., 2012; Ponnusamykonar et al., 2011). Keeping the above points in view, this research paper deals with the molecular confirmation of Trichoderma spp. evaluation of their metabolites and checking their efficacy individually and in combination under field conditions.

Survey and isolation of S. rolfsii

The survey was conducted in major groundnut growing regions like Thiruvannamalai, Sivagangai, Theni, Salem and Madurai districts of Tamil Nadu. The percentage of the disease incidence was calculated based on the severity of the infection. The stem rot pathogen was isolated through a tissue segmentation method (Kumar et al., 2014). Totally ten isolates were maintained (Table 1). Mycelium proliferated well within 2 days and produced sclerotia on 12^th day. The virulence of all the isolates of S.rolfsii was checked through pathogenicity test. It revealed that the isolate IS(BDI)-8 is the most virulent by recording 89.27 per cent incidence.



Isolation of Trichoderma spp.

During the survey, the soil samples were collected from the healthy groundnut rhizosphere region. Trichoderma spp. were isolated through soil dilution plating technique in Trichoderma selective medium (TSM) (Elad and Chet, 1983).

In-vitro screening of Trichoderma spp. against S. rolfsii 
 
Twenty five isolates of Trichoderma spp. were evaluated in vitro against S. rolfsii by  dual culture technique (Dennis and Webster, 1971). Seven days old cultures of Trichoderma spp. and S. rolfsii IS (BDI)-8 were used for the study. The culture disc (9 mm dia.) of the test pathogen and Trichoderma sp. were cut out and placed aseptically at equidistance and in opposite directions to each other on solidified PDA medium in Petri plates and then plates were incubated at 28±2^oC. Three replications were carried out. The PDA plates inoculated only with the culture disc of the test pathogen were served as control. The inhibition percentage of radial growth of the pathogen by Trichoderma sp. was compared with control (Vincent, 1947).
 
Per cent inhibition (I) = C - T/C × 100

Dc = Average diameter of fungal growth (cm) in control.
Dt = Average diameter of fungal growth (cm) in treatment.
 
Molecular confirmation
 
The most virulent S. rolfsii IS(BDI)-8 isolate and the elite Trichoderma spp. isolates T(AR)-10, T(SP)-20 chosen based on dual culture and were confirmed using molecular tool. DNA extraction was carried out using CTAB method. The reaction mixture (10 µl) for PCR amplification comprises of master mix (5 µl), forward and reverse primer (each 1 µl), DNA template (2 µl) and sterile water (1 µl). Master mix contains 0.25 mM dNTPs, 1.5 mM MgCl₂, Taq polymerase and buffer. The sequence of genomic DNA was amplified using the Forward primer: ITS 1(5'-TCCGTAGGTGAAC CTGCGG-3') and Reverse primer : ITS4 (5'-TCCTCCGCTTA TTGATATGC-3') PCR was done in a master cycler with inbuilt program of  initial denaturation at 94oC for 5 mins, continued by 35 cycles of denaturation  at 94^oC for 1 mins, annealing at 46^oC for 1 mins, extension at 72^oC for 1 mins and ended up after a final extension at 72^oC for 10 mins (White et al., 1990).The PCR products were subjected to gel electro phoresis on 1.5 per cent concentration. Then it was visualized and documented in gel documentation system.

The ITS gene sequences of the virulent S.rolfsii IS(BDI)-8 and proficient Trichoderma spp. T(SP)-20, T(AR)-10 were compared with NCBI blast search  gene bank data base (http://www.ncbi,nlm.nih.g). The sequences were submitted to the gene bank and received the accession numbers namely MZ277282-(Athelia rolfsii IS(BDI)-8), MZ277326-(Trichoderma asperellum T(AR)-10) and MZ277378-(Trichoderma longibrachiatum T(SP)-20).
 
Agar well diffusion method and GC-MS analysis

Trichoderma spp. isolates with highest antagonistic activity (Table 4) were further investigated for their ability to produce inhibitory metabolites. The Trichoderma sp. was grown in a conical flask containing PDA broth which was kept at 125 rpm and 28±2^oC in a shaker cum incubator for up to 7 days. After that, the content was centrifuged at 8000 rpm for 15 mins and the supernatant was filtered through Whatman no.1 filter paper to remove the residues of mycelia. Culture filtrate was extracted with ethyl acetate at the ratio of 1:1 and separated by a separation funnel. The extract was passed through anhydrous sodium sulfate to remove the excess water content. The extract was further concentrated in a rotary vacuum evaporator and used for agar well diffusion assay and GC/MS analysis. A nine mm mycelial disc of S. rolfsii was placed in the center of the Petri plate and then 100 µl of extract of effective Trichoderma sp. was dropped into the agar well 1 cm away from the edge at four sides on the periphery of Petri plate. The plates were incubated at room temperature and the plates were scored when the mycelium covered the entire Petri plate in control. Control was maintained with the sterile distilled water instead of crude extract.

Management of stem rot disease using elite Trichoderma spp. in field 

A field experiment was conducted in a stem rot affected groundnut field in Salem district of Tamil Nadu, India. The groundnut variety used was VRI-2 to evaluate the Trichoderma spp. for the management of stem rot. The field, which was abandoned for commercial cultivation due to severe stem rot incidence, was selected for this purpose. The talc formulation of effective Trichoderma isolates viz., T. longibrachiatum T(SP)-20 and T. asperellum T(AR)-10 was applied through seed treatment 4 g/kg of seed and soil application (@ 2.5 kg/ha). Three replications per treatment were maintained. The observations such as per cent disease incidence, per cent reduction over control and yield (kg/ha) were taken at the time of harvest.

Statistical analysis

Mean differences of the treatment were evaluated with ANOVA at a significant level (P<0.05) and means were compared by Duncan’s multiple range test (DMRT) (Gomez and Gomez, 1984).

Survey and isolation of stem rot pathogen

During the survey, highest (62.34%) and least (14.43%) stem rot incidence were recorded in Bodi of Theni district and Edappadi of Salem district (Table 1). The isolate IS (BDI)-8 showed maximum  incidence in groundnut field, grew very fast and produced maximum  number of sclerotia in the Petri plate (162 per plate). The color of the isolates ranged from dull white to pure white (Table 2,). Sivakumar et al. (2016) conducted a survey in different locations of groundnut fields at Cuddalore district. Among them Adhivaraganullur village registered the maximum incidence of 32.0% followed by Ponveli, (29.56%) and least disease incidence was recorded in Rajkuppam (7.88%) and the highly virulent isolate produced the light brown colored mycelium and 346 sclerotia per plate.


 
Isolation of Trichoderma spp. from different location of Tamil Nadu

The 25 different Trichoderma spp. were isolated and confirmed based on morphological characters such as color and growth pattern of mycelium, shape of the conidia and phialids (Rifai, 1969). The Isolate T(AR)-10  produced  green colored with ring-like zone of culture and  T(SP)-20 produced slight yellowish ring like growth pattern of culture in the Petri plate. 

Antagonistic activity of Trichoderma spp. against the stem rot pathogen

The Trichoderma sp. T(SP)-20 exhibited the maximum percentage inhibition (84.4%) followed by T(AR)-10 (75.5%) and TNAU TA (75.0%). (Table 3). Babu and Kumar (2008) reported that nine Trichoderma spp. (Th-1 to Th-9) were isolated from the microflora of the groundnut rhizosphere. Among them, the isolate Th-3inhibited the S. rolfsii mycelial growth up to 83% in the dual culture technique. Similarly, Hirpara et al. (2016) noticed that Trichoderma virens NBAII Tvs12 inhibited the mycelial growth of S. rolfsii  by coiling surround and production of hook like structure inside the mycelium of test pathogen and exhibited the percent growth reduction of 76.37% and 87.91% at 6 DAI and 12 DAI respectively and also arrested the sclerotia production compared to the control.



Molecular confirmation of Sclerotium rolfsii and Trichoderma spp.

The virulent isolate IS (BDI)-8 was amplified at the specific size of 700 bp using ITS1 and ITS4 primers depicting the molecular based confirmation of S. rolfsii. Similar results were obtained by Prasad et al. (2010). These workers performed rRNA amplification of S. rolfsii with ITS1 and ITS4 primers which produced a fragment of approximate size between 650 to 700 bp.

The best antagonists T(SP)-20 and T(AR)-10 chosen based on dual culture when amplified using ITS1 and ITS4 primers yielded a product of approximately 650-700 bp. According to Shahid (2013), universal primers (ITS-1 and ITS4) were used for the amplification of 28S rRNA gene of Trichoderma longibrachiatum and a sharp band of about 700 bp was seen on the gel.

Agar well diffusion assay

The culture filtrate extracts of six isolates (T(SP)-20, T(AR)-10, T(VT)-3,T(BI)- 16, T(TK)-23, TNAU-TA) were assessed against the S. rolfsii. Among them T(SP)-20 exhibited the maximum radial growth inhibition (1.1 cm) followed by T(AR)-10 (0.9 cm) (Table 4).



GC-MS analysis of Trichoderma spp.

According to GC-MS analysis it is clear that the T(SP)-20 and T(SP)-10 produced various antifungal compounds. The major antimicrobial compounds detected from T(SP)-20 of peak area% and RT value are Pentadecafluorooctanoic acid (1.29) (15.186), Quinoline,1,2-dihydro2,2,4trimethyl (1.97) (19.191), Pentadecane (1.48) (20.561), Nonadecane (6.75) (23.081), Hexadecanoicacid (3.07) (30.421, Phthalic acid (1.61) (31.101), 2-Trichosenoic acid (3.29) (36.655) (Table 5a).

The isolate T(AR)10 produced the antifungal compounds such as phenol (27.18) (6.819), 2, 6, 10 trimethyltridecane (3.44) (16.261), nonadecane (5.36) (23.077), 9-undeconal, 2, 10 dimethyl (3.18) (26.625), heptacosanoic acid (2.56) (30.407), acetic acid (2.51) (31.190), erucic acid (3.46) (38.880) (Table 5b, Fig 7b). Similarly, Siddiquee et al., (2012) reported that T. harzianum produced 278 volatile compounds like alkanes, alcohols, ketones, pyrones (lactones) etc., possessing the antifungal activity against a wide range of soil borne pathogens. Lee et al., (2016) noticed that Trichoderma sp.produced many volatile compounds such ascedrane, isobutyl acetate, caryophyllene, pentadecane, p-xylene, benzoic acid, pyridine, acetic acid and butanoic acid having the antimicrobial activity and induced the growth promotion in plants.





Management of stem rot disease using elite Trichoderma spp. in field 

The results depicted that all the modules consisting with Trichoderma sp. either alone or in combinations were found superior in reducing the stem rot disease incidence and resulted maximum pod yield as compared to control. The combined module treatment, T6 - ST with (T(SP)-20+T(AR)-10) @ 4 g/kg+SA of (T(SP)-20+T(AR)-10) @ 2.5 kg/ha before sowing+SA of (T(SP)-20+T(AR)-10) @ 2.5 kg/ha after 30 DAS (1:1) recorded the least disease incidence, highest disease control and pod yield (9.22%, 82.67% and 1452 kg ha^-1) respectively followed by T4 - ST with T(SP)-20 @ 4 g/kg + SA of T(SP)-20 @ 2.5 kg/ha before sowing + SA of T(SP)-20 @ 2.5 kg/ha after 30 DAS (11.22%,78.91% and 1370 kg ha^-1) (Table 6). Conclusively, the combined application of two Trichoderma spp. used as seed treatment and soil application reduced the disease incidence more effectively as compared to seed treatment and soil application of Trichoderma sp. alone. The Trichoderma sp. treatments significantly enhanced the yield attributes and reduced the disease incidence. Similarly, Meena et al. (2018) reported that soil application of T. harzianum (Th-BKN) @ 10 kg ha^-1 +FYM @ 10 tonnes.ha^-1 gave highest disease control, pod yield in kg ha^-1 and lowest disease incidence was 86.30%, 2173 kg ha^-1 and 7.51% respectively.

From this experimental result, the Trichoderma longibrachiatum T(SP)-20, Trichoderma asperellum T(AR)-10 produced numerous organic compounds with antimicrobial activity against a wide range of pathogens including S. rolfsii. Combined talc-based application of the above two species through seed treatment and soil application is found highly effective reducing the stem rot incidence compared to individual application. In near future, commercially it will be exploited for the management of stem rot of groundnut.