Unveiling the potential of Trichoderma harzianum against Heterodera cajani in pigeon pea: impact on cysts, egg and juveniles abundance

Background Pigeon pea is affected by the cyst nematode Heterodera cajani limiting the production. H. cajani can be managed by biological agents such as Trichoderma harzianum . Thus, the determination of this experiment was to estimate native strains of T. harzianum isolated from pigeon pea fields in India on population of cyst, eggs and juveniles of H. cajani . Pots experiment was conducted at the playhouse of Sam Higginbottom University of Science and Technology, Prayagraj (UP), India, during the year of 2018–2020. Earthen pots were filled with sterilized soil @ 10 kg/ pot for each replicate of the treatments. Ten days before sowing of the pigeon pea seed, about 5000 spores of T. harzianum were amended in to the soil for proper colonization. Fifteen days after seed germination, 200 cysts/pot were inoculated near the root zone of pigeon pea plant. The pots were irrigated when required. Observations were recorded of cyst population, eggs and second juveniles of H. cajani /500gm of pigeon pea rhizospheric soil at 90 days after sowing. Results Among all the isolates of T. harzianum , the treatment (T 7) reduced of cyst population (12), eggs population (234) and juveniles’ population (153) as compared with all other treatments including control at 90 days after sowing of pigeon pea seeds in the year 2018–2019. Similarly, in 2019–2020 reduction in cyst population (11), eggs population (189) and juveniles population (160) was observed at 90 days after sowing in Trichoderma isolates (T 7) as compared to control. Conclusions The findings of this study are very relevant since seven isolates of native T. harzianum were promising to suppress the number of cysts in 79.31 and 83.07%, eggs in 22.00 and 38.83% and juveniles’ population in 17.74 and 20.39% of the nematode H. cajani, besides promoting the pigeon pea plant growth.


Background
The pigeon pea (Cajanus cajan L.), which ranked sixth internationally behind peas, broad beans, lentils, chickpeas and common beans, is farmed on 5.4 million hectares of land worldwide and produces 4.49 million metric tons annually (Fatokimi and Tanimonure 2021).More than 100 pathogens attack this crop including nematodes, fungus, viruses, bacteria and MLOs (Reddy 2021).Significant damage to the crop at all stages is caused by plant parasitic nematodes which are microscopic (Maurya et al. 2020a).Pest causes up to 50% of potential crop loss, while 12.3% is supposed to be caused by nematodes and more damage to the crops due to nematode infestation is detected in the emerging countries than in the industrialized countries (Carneiro et al. 2017).Scientists have become interested in plant parasitic nematodes since they have been linked to significant, widespread diseases.There are many genera and species of plant parasitic nematodes that reduce the quality and quantity of yields in different crops while also raising the price of production.The crop of pigeon peas suffers significant damage from plant parasitic nematodes, which causes the plants' physiological processes to malfunction, lowering crop yield.The cyst nematode H. cajani has been shown to live on pigeon pea, and this parasite is by far the most prevalent (Briar et al. 2023).According to Ali and Askary (2001), H. cajani is among the most significant pests of pigeon pea in India, along with M. incognita, M. javanica and R. reniformis.According to Abd-Elgawad and Askary (2015), plant parasitic nematodes are responsible for a 13.2% yearly output loss in pigeon pea production globally.Adult females of H. cajani can be identified by their lemon-shaped forms, short necks and terminal cones.The cysts initially have a light yellow tint, but over time, they form strong walls, progress from yellow to brown to black, and eventually turn entirely black.H. cajani females are very different from females of other species.Their huge egg sac, which can occasionally be nearly twice as big as the cyst itself, is a distinguishing feature of these growths (Ali and Singh 2005).Edward and Misra (1968) reported H. vigni from the roots of pigeon pea (Vigna unguiculata), which was later discovered to be synonymous with H. cajani.
The use of Trichoderma harzianum as a biological control has been very effective in sustainable agricultural systems.The fungus is effective in controlling nematodes such as Heterodera (genera) by colonizing and trapping them (Tylka and Marett 2022), reducing disease through the predation of nematodes (Maina et al. 2022), its nematotoxic effects (Mhatre et al. 2022) and inhibiting egg hatching and juvenile mortality (Kumar et al. 2020).Therefore, the purpose of this investigation was to assess the effects of local T. harzianum strains found in Indian pigeon pea fields on the population of cysts, eggs and juveniles of Heterodera cajani.

Trichoderma harzianum-isolation, purification and identification
The process of isolation of T. harzianum was made from the rhizosphere of a healthy pigeon pea plant in the region of Prayagraj, UP in 10 different fields.The soil in the 10-cm rhizosphere was carefully scraped out from around the roots, which were then sliced into little pieces and mixed with the soil.T. harzianum were isolated using the serial dilution plate method (Sharma et al. 2019).The conidium morphology of T. harzianum can be used to quickly identify them.This species can be distinguished from all other T. harzianum by its small, subglobose conidia, which are typically 2-3 to 1.5-2.5 m in size.Conidiophores have a pyramidal, verticillate branching structure and lack a sterile apical elongation at maturity, despite the fact that developing conidiophores may show a visible sterile extension in the early stages of growth.

Collection, processed and identification of cyst nematode from infested soil
Infected soil was collected from different fields and processed by sieve methods.In this processing, 500 g of infested soil was passed through 25 mesh and then 60 mesh, the cyst of Heterodera (Da Silva Café and Santos 2023) cajani cannot pass through 60 mesh thus were collected carefully.The nematode suspension was stirred and poured in a small quantity in the counting dish.The counting dish was rotated under the low-power binocular microscope in search of cyst nematode.When the cyst appeared into the field, they were identified.The cysts were counted by using the counter and collected into the cavity block with the help of forceps (Maurya et al. 2022b) (Fig. 1).The female cyst is lemon shaped.The cuticle is marked by thick, zigzag lines.One large and one smaller annule is present on the head region (Koshy 1967).Cowpea seeds were sown for the mass multiplication of H. cajani in pot (Fig. 2).

Experimental site
Pots experiment was conducted at the playhouse of Sam Higginbottom University of Science and Technology, Prayagraj (UP), India, during the year of 2018-2020.Prayagraj was 98 masl and was sited at 25.27 levels north and 81.50 ranged east.Commonly, the climate is subtropical and semiarid.In the summer season, the maximum temperature might also get as excessive as 46.5 °C, and in the winter, it can reach up to 1.5 °C.Approximately, 1100 mm of rain precipitation takes place annually on common.

Preparation of treatments
The earthen pots were used for the experiment.The experiment's pots were washed in running water.The soil was collected and sterilized with 1% formalin solutions, earthen pots were filled with sterilized sandy loam soil @ 10 kg/pot for each replicate of the treatments.Before sowing the seed, about 5000 spores of T. harzianum (calculated by hemocytometer) were amended into the 10 kg of soil for proper colonization.Ten days after amendments of T. harzianum, seeds of pigeon pea (Upaas variety) were sown in each pot.In control pots, soil without amendment and one chemical (Carbofuran) were maintained for comparison.Fifteen days after seed germination, 200 cysts/ pot were inoculated near the root zone of pigeon pea plant.The pots were irrigated when required.Observations were recorded of cyst population, eggs and second juveniles of H. cajani (Min et al. 2020)/500gm of pigeon pea rhizospheric soil at 90 days after sowing.

Data analysis
A completely randomized design (CRD) was employed as the experimental design with three replications.A design of freedom (df ) that was appropriate was created using the calculated value of F at a 5% level of probability (Chandel 2010;Abd-Elgawad 2021).Data expressed as percentages were transformed according to the angular transformation.

Table 1
Evaluation the isolates of Trichoderma harzianum on cyst population, eggs and juveniles of Heterodera cajani/500 g of pigeon pea rhizospheric soil(2018)(2019)

Table 2
Evaluation the isolates of Trichoderma harzianum on cyst population, eggs juveniles of Heterodera cajani/500 g of pigeon pea rhizospheric soil(2019)(2020) Fig.4Evaluation the isolates of Trichoderma harzianum on cyst population, eggs and juveniles of Heterodera cajani/500 g of pigeon pea rhizospheric soil(2019)(2020) (Mhatre et al. 2022)7)orted that maximum reduction in cyst nematode (H.cajani) multiplication was found in pigeon pea when G. mosseae, T. harzianum and P. chlamydosporia were used together.Use of T. viride reduced by the incidence of H. cajani on green gram, red gram and black gram(Alase et al. 2020).According toCarneiro et al. (2017), Trichoderma spp.had also been shown to colonize Globodera rostochiensis eggs and juveniles and devour the egg contents(Krif et al. 2020).The bioagents Fusarium spp., Trichoderma spp., B. thuringiensis and Saccharopolyspora spinosa may cause a drop in nematode levels at the rates utilized in this experiment and may serve as a replacement control alternative to suppress Meloidogyne sp., G. rostochiensis.G. rostochiensis' growth was significantly inhibited by Trichoderma species.Reproduction rates dropped by 36.0-44.4% for both fungi and bacterial preparations by 27.7-33.3%thanthecontrols(Mhatreet al. 2022).The findings of this study are very relevant since seven isolates of native T. harzianum are promising candidates to suppress the number of cysts, eggs and juveniles population of the phytopathogen nematode H. cajani, in addition to promote plant growth.Seven isolates of the fungus T. harzianum are promising candidates to reduce the population of the nematode H. cajani's juveniles by 17.74 and 20.39%, eggs by 22.00 and 38.83%, and cysts by 79.31 and 83.07%.The level of 5000T.harzianum spores in 10 kg of soil had the capacity to reduce the number of H. cajani cysts, eggs and juveniles.The pigeon pea plant can grow more quickly thanks to T. harzianum.These local Trichoderma isolates can be regularly employed to control plant pathogenic nematodes that harm legumes.