Introduction

The utilization of green gram (Vigna radiata L.) Wilczek in Pakistan as diet has been expanding for the last few years as a substitute of protein and hence, has been distinguished as high need crop by the farmers. Along these lines, expanding the productivity of green gram has been recognized as a basic need to meet the nation’s requirements and guarantee self-sufficiency. Increasing the production of green gram would result in uplifting the income level of farmers and meeting dietary needs of the people of the nation. Green gram is cultivated on an area of 246 thousand hectares with a total production of 178 thousand tons respectively (Anonymous 2018) under a wide range of agro-ecological zones. The normal yield of green gram in Pakistan is far below than its potential yield. Several biotic and abiotic factors affect the yield of green gram (Aslam et al. 2019a; Mukhtar and Hussain 2019). Among biotic factors, diseases are the most destructive. Green gram is attacked by more than 26 diseases throughout the world reducing yield up to 44% (Iqbal and Mukhtar 2020a). Among diseases, root-knot nematodes (Meloidogyne spp.) are by far the most destructive and badly impact crop production. Javanese root-knot nematode, Meloidogyne javanica, has been found widely distributed infecting a wide range of crops in the country (Trudgill and Blok 2001; Castagnone-Sereno et al. 2013; Jones et al. 2013; Mukhtar and Kayani 2019, 2020; Tariq-Khan et al. 2020). Root-knot nematodes have been reported to inflict yield losses in pulses to the extent of 26%. Root-knot nematodes are ubiquitously prevalent in tropical and subtropical environments of the country as prevailing climatic conditions are conducive for their pathogenesis and reproduction throughout the year (Hussain and Mukhtar 2019). Furthermore, these nematodes develop disease complexes with soil borne fungal and bacterial pathogens causing vascular wilts and damping off in many crops and result in huge yield losses. Similarly, root-knot nematodes break resistance in cultivars resistant to wilts (Aslam et al. 2019b; Asghar et al. 2020).

For the management of nematodes, various control methods are used which include resistance, chemical, biological, physical, regulatory and cultural etc. (Bhuiyan et al. 2018; d’Errico et al. 2019; Javed et al. 28,29,a, b; Nazir et al. 2019; Azeem et al. 2021; Gulzar et al. 2020) and each strategy has one or the other limitations. Root-knot nematodes are mainly controlled by using synthetic nematicides, but their excessive use is prohibited due to non-availability of conventional nematicides, growing awareness of global community against pollution, possible health hazards associated with the use of nematicides, unfavorable cost-benefit ratio, and development of resistance in nematodes etc. which demands for suitable alternatives. The overreliance on nematicides can be curtailed by using biological control agents possessing nematicidal or nematostatic properties.

Several bacterial and fungal biological control agents have been evaluated during recent years for their effectiveness against root-knot nematodes (Huang et al. 2016; Rao et al. 2017; Ghahremani et al. 2019). The main criteria for successful deployment of biocontrol agents in fields are their ability to suppress nematode populations and enhance yields profitably in the presence of nematodes. For their suitability as nematode suppressive agents, the reductions in reproductive and developmental potentials of nematodes by these biocontrol agents must be assessed. Among different groups of fungi, the genus Trichoderma, as biocontrol agent of different plant pathogens, has been widely studied. Its species are often very fast growing, colonizing the substrates quickly. The fungus is ubiquitous and has been reported to effectively control different diseases by using a variety of mechanisms (Vinale et al. 2008; Ghazanfar et al. 2018). Aggressiveness among various species and isolates of Trichoderma from different regions of the world has been known (Mukhtar 2018; Iqbal and Mukhtar 2020b). This necessitates that indigenous isolates of the fungus should be used for the management of root-knot nematodes. There is meagre cognizance on the effects of indigenous isolates of Trichoderma species on the reproductive potential of nematodes and growth variables of green gram. In the present study, effectiveness of seven indigenous species of Trichoderma were tested for their ability to suppress population of Javanese root-knot nematode, M. javanica, and improve growth variables of green gram.

Materials and Methods

Nematode Inoculum

A population of Javanese root-knot nematode (Meloidogyne javanica) originally isolated from cucumber roots, identified on the basis of perineal pattern and maintained on highly susceptible cultivar of tomato (Money Maker) was used in the assessment. The nematode was mass produced on tomato cv. Money Maker and second stage juveniles were extracted from the infected roots for inoculation of plants (Khan et al. 2019).

Mass Production of Trichoderma Species

The seven species of Trichoderma evaluated in the present study were obtained from the Institute of Agricultural Sciences, University of Punjab, Lahore. These species, originally isolated from the country, identified and confirmed were maintained in the Fungal Culture Bank of the university. The species included Trichoderma harzianum, T. hamatum, T. koningii, T. pseudokoningii, T. viride, T. atroviride and T. asperellum. These biocontrol fungi were first grown on potato dextrose agar and then mass produced individually on wheat grains using the methodology described by Mukhtar (2018). The concentration of spores per gram of the grains of each Trichoderma spp. was counted using haemocytometer after making spore suspensions in distilled water.

Greenhouse Bioassay for the Effectiveness of Trichoderma Species Against M. javanica

The relative effectiveness of seven Trichoderma species against M. javanica was assessed in pots under greenhouse conditions. Each Trichoderma spp. colonized on wheat grains was mixed with formalin sterilized soil (60% sand; 19% silt; 20% clay; 1% organic matter and pH 7.6) at the rate of 104 spores/ml and filled in pots separately. The pots treated with wheat grains only served as controls. Three seeds of green gram (cv. AZRI-06) were sown in pots treated with Trichoderma species. Each treatment was replicated five times. The pots were placed in a greenhouse at 25 ± 2 °C in completely randomized design. After emergence, single healthy seedling was maintained in each pot. Each plant was inoculated ten days after emergence with approximately 2000 freshly hatched second stage juveniles of M. javanica contained in 2 ml of water by making three holes around the stem. The pots were kept for seven weeks in the greenhouse at 25 ± 2 °C and watered as per requirement.

Data Recording

After seven weeks, the plants were carefully removed from the pots. The shoots were severed from the roots. Shoot and root lengths were measured, and fresh root and shoot weights were determined. The galls and egg masses on each plant root system were counted under a stereomicroscope (NIKON SMZ745T) at 40×. For estimation of total nematode populations, eggs were extracted from the roots of individual plants and juveniles were extracted from the soil from each pot (Mukhtar 2018). The total number of eggs and nematodes in soil formed the total population. The reproductive factor was calculated by dividing the final population by the initial one. Percent reductions or increases in plant growth parameters and nematode infestations were calculated over controls as described by Mukhtar (2018).

Statistical Analysis

Completely randomized design was used in the experiment. All the data were subjected to analysis of variance using GenStat Package 2009 (12th edition) version 12.1.0.3278 (www.vsni.co.uk). Means were compared by Fisher’s Protected Least Significant Difference Test at 5%.

Results

The analysis of variance showed significant results regarding effects of Trichoderma species on growth variables and nematode infestations. All the Trichoderma species resulted in an increase in shoot and root lengths and shoot weight while a decrease was observed in root weight. Maximum increase in shoot length was found to be maximum in case of T. harzianum (45.5%) followed by T. hamatum (44.4%) and T. viride (42.3%) whereas the increase was the minimum where T. pseudokoningii and T. koningii were applied (Fig. 1). Similarly, maximum increase in shoot weight was recorded with T. viride (56.1%) followed by T. harzianum (55%) and the minimum with T. pseudokoningii (Fig. 2). As regards root length, it was the maximum in treatments with T. hamatum (46.2%) and T. harzianum (45.1%) and minimum with those where T. koningii and T. pseudokoningii were applied (Fig. 3). Contrarily, maximum reduction in root weight was observed in treatments where T. harzianum (37.8%) and T. viride (35.8%) were applied while T. koningii and T. pseudokoningii resulted in the minimum decrease (Fig. 4). All the Trichoderma species significantly caused reductions in number of galls and eggmasses and reproductive factor of the nematode over control. Maximum reduction in number of galls and eggmasses was observed with T. viride (49 and 53%) followed by T. harzianum (46 and 53%) and T. hamatum (49%) while the minimum reduction was recorded with T. pseudokoningii followed by T. atroviride (Figs. 5 and 6). Likewise, T. viride caused the maximum reduction in reproductive factor of M. javanica (81%) followed by T. harzianum (78%) and T. asperellum (75%). On the other hand, the minimum reduction in reproductive factor was observed with T. pseudokoningii and T. koningii (Fig. 7).

Fig. 1
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Effect of Trichoderma species on percent increase in shoot length of green gram

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Fig. 2
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Effect of Trichoderma species on percent increase in shoot weight of green gram

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Fig. 3
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Effect of Trichoderma species on percent increase in root length of green gram

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Fig. 4
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Effect of Trichoderma species on percent decrease in root weight of green gram

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Fig. 5
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Effect of Trichoderma species on percent decrease in number of galls on roots of green gram

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Fig. 6
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Effect of Trichoderma species on percent decrease in number of eggmasses on roots of green gram

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Fig. 7
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Effect of Trichoderma species on percent decrease in reproductive factor of Meloidogyne javanica

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Discussion

Trichoderma species are free-living soil borne fungi and are commonly found in root ecosystems. These opportunistic fungi develop symbiotic relationships with different kinds of plants and fungi. Some species of Trichoderma have the ability to colonize root surfaces and enter the epidermis resultantly enhancing development and growth of roots, increasing production, imparting resistance to abiotic stresses and uptake of nutrients is greatly improved. In the present study, effectiveness of seven indigenous species of Trichoderma was tested for their ability to suppress population of Javanese root-knot nematode and improve growth variables of green gram. All the Trichoderma species resulted in increase in plant growth and caused reductions in nematode infestations over control.

Trichoderma species have been found effective against several phytopathogenic nematodes and fungi. Several species of Trichoderma including T. harzianum, T. viride, T. atroviride, T. citrinoviride and T. asperellum have provided excellent control of root-knot nematodes in previous studies (Sharon et al. 2007; Affokpon et al. 2011; Saikia et al. 2013; Martínez-Medina et al. 2017; Sonkar et al. 2018). In one study, T. harzianum and T. viride reduced root galling by M. incognita and M. javanica by 31% in tomato (Sharon et al. 2001; Dababat et al. 2006). Bokhari (2009) showed that T. harzianum, T. hamatum, T. koningii, T. viride and T. reesei culture filtrates significantly decreased females and eggs of reniform and root-knot nematodes. Jindapunnapat et al. (2013) found that inoculation of root zones of guava plants with T. harzianum reduced nematode numbers in soil and roots and arrested the development of juveniles. Similarly, another species T. longibrachiatum significantly controlled M. incognita on cucumber (Zhang et al. 2015). Al-Hazmi and Javeed (2016) reported that T. harzianum and T. viride suppressed nematode multiplication and root galling and increased growth of tomato plants. Moreover, many soil borne pathogens including Rhizoctonia solani, Pythium ultimum, Fusarium moniliforme, Macrophomina phaseolina and Sclerotium rolfsii have also been controlled by different Trichoderma species (Bae et al. 2016; Giurgiu et al. 2018; Ghazanfar et al. 2018; Iqbal and Mukhtar 2020b).

Several mechanisms have been reported to be involved in the biocontrol activity of Trichoderma against fungal plant pathogens. The important mechanisms are antibiosis, competition, enzymatic hydrolysis, parasitism and systemic induced resistance (Benitez et al. 2004; Harman et al. 2004; Ragozzino and d’Errico 2011; Hhmau et al. 2015; Lombardi et al. 2018). Many biocontrol compounds and activators such as Trichokonins, trichodermin and trypsin-like protease obtained from Trichoderma spp. have been proven to be nematicidal (Suarez et al. 2004; Xiao-Yan et al. 2006; Reino et al. 2008; Chen et al. 2009; Yang et al. 2010, 2012). The highly branched conidiophores of Trichoderma produce conidia that can attach to different nematode stages. Conidial attachment and parasitism varies among fungal species and strains (Sharon et al. 2007). This process was often associated with the formation of fungal coiling and appressorium-like structures. T. harzianum colonizes isolated eggs and J2s of M. javanica (Sharon et al. 2007). Successful parasitism of the nematode by Trichoderma requires mechanisms to facilitate penetration of the nematode cuticles or eggshells. The involvement of lytic enzymes has long been suggested and demonstrated in Meloidogyne parasitism (Spiegel et al. 2005). Besides direct antagonism, other mechanisms involved in Meloidogyne control by Trichoderma spp. include production of fungal metabolites and induced resistance (Bokhari 2009). Reduction in nematode galls and eggs might be due to high rhizosphere competency of bio-agents as they can easily colonize roots and may reduce feeding sites for nematodes. The reduction in root galling might be due to failure of majority of the juveniles to enter the host roots. In general, Trichoderma should be applied before planting to achieve maximum nematode control as good establishment of the fungus in plant rhizospheres seems to be important for nematode control.

In the present study, Trichoderma species significantly enhanced growth parameters of green gram over control. Plant growth enhancement by Trichoderma isolates can be attributed to different mechanisms which include enhanced nutrient uptake, solubilization of phosphates, sequestration of inorganic nutrients, secretion of exogenous enzymes, siderophores and enhanced root hair development (Harman 2006; Lorito et al. 2010). Trichoderma was found helpful in increasing plant hormones which help to increase root hairs and growth and make them more efficient in absorbing nitrogen, phosphorus, potassium and micronutrient (Mastouri et al. 2010). Besides enhanced growth and yields, Trichoderma species also increased chlorophyll, starch, nucleic acids contents, total protein and phytohormones in maize plants (Akladious and Abbas 2014). In the present study, Trichoderma species showed variations in their effectiveness as growth promotor of green gram and in suppressing nematode. This might be due to variations in genetics, pathogenic potential and the origins of different Trichoderma species and isolates.

Conclusion

All the seven Trichoderma species showed nematicidal potential against Meloidogyne javanica and caused significant reductions in nematode infestations resulting in enhancement of growth variables of green gram. Although Trichoderma species varied in their effectiveness against the nematode but Trichoderma viride, T. harzianum, T. asperellum and T. hamatum proved more effective and can be potential alternatives to synthetic nematicides. As these Trichoderma species are commonly found in the country they can be effectively exploited for the management of root-knot nematodes.