Background

Cowpea (Vigna unguiculata L.) is one of the most important, oldest, herbaceous legume crop, widely cultivated for fodder and grain in the Pakistan and semi-arid tropics of the world (Mensack et al. 2010). Its substantial adaptation to drought, elevated temperatures, a wider spectrum of pH, requirement of less fertilizers and minimal irrigation relative to many other legumes, increase its preference by the farmers in improving their socio-economic status and in contributing agricultural productivity. However, cowpea growth and productivity are suppressed by very destructive and economically important charcoal rot disease caused by the seed and soil-borne necrotrophic fungus Macrophomina phaseolina (Tassi) Goid. The pathogen is widely distributed in the regions with high temperatures and drought conditions, while it is responsible for infecting more than 500 plant species including cowpea, mung bean, chickpea, sorghum, sunflower, etc. Disease causes wilting of host plant after infection and pathogen keeps on producing microsclerotia in senescing shoot tissues which causes further decay of host tissue (Mayek-Perez et al. 2001).

There are no effective fungicides or other control methods to limit M. phaseolina (Gaige et al. 2010). Disease management through utilizing native antagonistic soil fungi and allelopathic plants is an attractive alternative among the disease management practices. Trichoderma is a common filamentous biocontrol fungal agent, found almost in any soil type. The antifungal activity of this genus is associated with improvement in growth and systemic resistance in plant (Harman et al. 2006). Several antagonistic mechanisms like nutrient competition, antibiotic production, and mycoparasitism generally work in Trichoderma against the pathogen (Vinale et al. 2008). Many biocontrol mycoparasitic species of Trichoderma have been well studied including T. harzianum, T. viride, T. hamatum, T. koningii, and T. reesei against M. phaseolina (Khalili et al. 2012), and many have been developed into a commercial biocontrol product.

Utilization of plant extract and biomass is another environment friendly way of managing the disease as a source of natural pesticides. Plants are store house of biochemicals that contribute in suppressing phytopathogens (Sales et al. 2016). These biochemicals (nitrogen-containing compounds and phenolics) function as a defense and chemical signal molecule against pathogens. Previous literature showed that phytochemicals of Melia azedarach and Azadirachta indica, besides holding medicinal values, have shown considerable fungicidal activity against pathogenic fungi including M. phaseolina (Carpinella et al. 2003). The present study was planned to investigate antifungal activity of three indigenous Ascomycetes fungal species viz., T. harzianum, T. viride, and T. hamatum, and two Meliaceae members, i.e., M. azedarach and A. indica against M. phaseolina responsible for charcoal rot disease in cowpea through in vitro trials.

Materials and methods

Laboratory bioassays

Three Trichoderma species, i.e., T. viride (FCBP 644), T. harzianum (FCBP 1277), and T. hamatum (FCBP 907), were tested for their antagonism activity against M. phaseolina (FCBP 0751).

Antifungal activity of Trichoderma spp. by cell-free culture filtrates

Cell-free culture filtrates Trichoderma spp. were prepared in 2% ME (malt extract) broth medium (100 mL). After 20 days of inoculation, cell-free supernatants were collected after aseptic filtration through Whatman filter paper and centrifugation at 4000 rpm for 5 min, followed by re-filtration through Millipore filter paper (pore size 45 μm). Twenty-one different concentrations, ranging from 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,…, 100% (v/v) of each cell-free culture filtrate, were prepared by addition of 2% of ME. Flasks were inoculated with 5 mm disc of M. phaseolina and incubated at 28 ± 2 °C. After 7 days, mycelial mat was dried in oven at 45 °C for 24 h for measuring dry biomass.

Assessment of the antifungal potential of plant extract

Two hundred-gram powdered leaves of A. indica and M. azedarach were soaked in 2 L methanol separately for 12 days. Extracts were obtained from soaking materials by filtering and evaporating and finally drying. Original concentration was made by dissolving 9 g of extracting plant material in 5 ml of dimethyl sulphoxide (DMSO 99.5%) to prepare a final volume of 15 ml. Control solution was made by adding 5 ml of DMSO in 10 ml of sterilized distilled water. Six concentrations, i.e., 0, 1, 2, 3, 4, and 5%, were made by adding 0, 1, 2, 3, 4, and 5 ml of stock solution and 5, 4, 3, 2, 1, and 0 ml of control solution in 55 ml of each flask to make a final volume of medium 60 ml. Then, 60 ml of each treatment was equally divided into four 100-ml flasks to serve as replicates, where 0% was control treatment. Actively growing culture of M. phaseolina (5 mm disc) was inoculated in each flask and incubated at 28 ± 2 °C for 7 days. The fungal biomass was dried and weighed.

Pot bioassays

On the basis of the laboratory bioassays, two species of Trichoderma viz. T. harzianum and T. viride, and one member of Meliacaceae family, i.e., A. indica, were selected to conduct trials in pots (6 in. diameter × 10 in. height). Initially, presterilized potted soil (1 kg pot−1) was inoculated with cultural suspension (conidial count 4 × 106) of each of two Trichoderma spp. and left for 4 days for the establishment of the fungus in soil. Later dry leaves of A. indica were mixed at 1, 2, and 3% in 1 kg of soil and left for 7 days. Soil was inoculated with M. phaseolina (MP) and left for another 4 days for inoculum establishment. Finally, surface sterilized seeds of cowpea with 0.1% sodium hypochlorite solution were sown in each pot. The pots were arranged in a completely randomized design and were kept under natural environmental conditions having three replicates of each treatment. Experiment was comprised of 13 treatments including T 1 : negative control (without any inoculation or amendment); T 2 : positive control (inoculated with MP only); T 3 –T 5 : MP + 1% A. indica, MP + 2% A. indica and MP + 3% A. indica; T 6 : MP + T. harzianum; T 7 –T 9 : MP + T. harzianum + 1% A. indica, MP + T. harzianum + 2% A. indica, MP + T. harzianum + 3% A. indica; T 10 : MP + T. viride; T 11 –T 13 : MP + T. viride + 1% A. indica; MP + T. viride + 2% A. indica, and MP + T. viride + 3% A. indica.

Disease assessment

After 40 days of inoculation charcoal rot disease symptoms on cowpea plants, were appeared and were assessed, using disease rating scale, where 1: no symptoms on plants (highly resistant); 3: lesions are limited to cotyledonary tissues (resistant); 5: lesions have progressed from cotyledons to about 2 cm of stem tissues (tolerant); 7: lesions are extensive on stem and branches (susceptible); and 9: most of the stem and growing points are infected. A considerable amount of pycnidia and seclerotia are produced (highly susceptible). Disease incidence (DI) was determined, using the following formula:

$$ \mathrm{DI}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{infected}\ \mathrm{plants}}{\mathrm{Total}\ \mathrm{nutmber}\ \mathrm{of}\ \mathrm{plants}}\times 100 $$

Analysis of plant physiology

Physiological variations in different treatments were assessed in cowpea leaves after 40 days of seed sowing. Total chlorophyll content was quantitatively analyzed by taking absorbance properties for chlorophyll a (645 nm), chlorophyll b (663 nm), and carotenoid (270 nm), and the amount of pigment was calculated. Activity of catalase (CAT) was determined in the reaction mixture consisted enzyme extract (0.1 ml) that was added to 2.9 ml of H2O2 (20 mM) and sodium phosphate buffer (50 mol/L; pH 7.0) by monitoring the reduction in the absorbance at 240 nm (Maehly and Chance 1967). Activity of peroxidase (POX) was determined by taking 0.5 ml of enzyme extract in reaction mixture containing 2 ml of 0.1 mol/L phosphate buffer (pH 6.8) and 1 ml of pyrogallol. Solution was filled with 1 ml of 0.05 mol/L H2O2 (5:5 in H2O2 and distilled water), incubated at 25 °C, and reaction was stopped by adding 2.5 mol/L H2SO4 (24.5 ml of H2SO4 + 100 ml of distilled water). The amount of purpurogalline formed was determined by reading the absorbance at 430 nm against a blank prepared by adding the extract after the addition of 2.5 mol/L H2SO4 (Colville and Smirnoff 2008). Polyphenol oxidase activity (PPO) was assayed in a reaction mixture consisted of 0.1 ml enzyme extract and 1.5 ml of 0.1 mol/L sodium phosphate buffer (pH 7.0), 0.2 ml of 0.01 mol/L catechol. The changes in the absorbance were recorded at 30-s interval for 3 min at 495 nm (Mayer et al. 1965). For determination of phenylalanine ammonia-lyase (PAL) activity, reaction mixture [(0.4 ml of enzyme extract + 0.1 mol/L sodium borate buffer (pH 8.8) + 0.5 ml of 0.012 mol/L l-phenylalanine)] was incubated for 1 h in light at 25 °C and reaction was stopped by incubating at 47 °C for 10 min. The amount of trans-cinnamic acid formed was calculated after measuring absorbance of samples at 290 nm (Dickerson et al. 1984).

Harvesting and data collection

Plants were harvested after 65 days of sowing. Data regarding disease incidence, and plant height, shoot, root fresh, and dry weight were measured. Materials were dried at 70 °C, and dry weight was recorded on an electric balance.

Statistical analysis

Triplicate values reported are mean of ±SD. Kolmogorov-Smirnov test (normality test) and Levene’s test (variance test) were applied before any statistical analysis. When all the assumptions of ANOVA were satisfied, standard errors of all data were analyzed by analysis of variance (ANOVA), followed by LSD test, using computer software Statistix 8.1.

Results and discussion

Laboratory trials

Increasing concentrations (5–100%) of cell-free culture filtrate of the three Trichoderma spp. were found highly effective in suppressing growth of M. phaseolina. Thus, the highest inhibition of 10–90% in the biomass of M. phaseolina was recorded due to cell-free culture filtrate (CFC) of T. harzianum, followed by 5–70% due to CFC of both T. viride and T. hamatum as compared to control (Table 1). Likewise, Naglot et al. (2015) and Khaledi and Taheri (2016) reported inhibition in growth of M. phaseolina by different Trichoderma spp, whereas the difference in concentration of volatile substances (acetaldehyde, isocyanide derivatives, terpene, hydrazine, alcohols, lactones, etc.) and cell wall degrading enzymes (chitinase and glucanase) might be ascribed to dissimilar fungicidal activity of three Trichoderma spp. (Woo et al. 2006). Considering the significant antifungal activity of T. harzianum and T. viride against M. phaseolina, the two species were later used in the pot study.

Table 1 Effect of different concentrations of Trichoderma species filtrate on biomass (g) of Macrophomina phaseolina
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In laboratory trials, different concentrations (1–5%) of leaf extract of M. azedarach and A. indica significantly reduced M. phaseolina growth by 19–61 and 25–72%, respectively (Table 2). Reduction in fungal biomass to increase in the concentration of plant extract has been reported by several authors (Latha et al. 2009). Many chemicals and biological active compounds have been identified in the leaf extract of A. indica (phytol, octadecatrienoic acid, methyl ester, hexadecanoic acid, methyl ester, etc.) (Hossain et al. 2013) and in M. azedarach (β-sitosterol, β-amyrin, ursolic acid, benzoic acid, and 3-5 dimethoxy benzoic acid) (Jabeen et al. 2011). The significant reduction in growth of the fungus treated with two plant species was probably due to a difference in occurrence of inhibitors to the fungitoxic principle (Baka 2010).

Table 2 Effect of methanolic extract of Melia azadarch and Azadirachta indica concentrations on biomass (g) of Macrophomina phaseolina
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Pot trials

Effect on disease and growth

There was no disease in negative control. The highest disease incidence of 75% was recorded in positive control, where only M. phaseolina was inoculated in the soil. Soil amendments with 1–3% dry leaf biomass of A. indica significantly reduced the disease incidence from 50 to 30% over positive control. MP + T. harzianum or MP + T. viride significantly reduced disease incidence to 23 and 39%, respectively. The combined effect of antagonistic fungi with soil amendment was more pronounced. Therefore, T. harzianum with 1–3% dry biomass of leaf showed the highest reduction in disease incidence from 20 to 8% (Table 3).

Table 3 Effect of Macrophomina phaseolina, soil amendment, and Trichoderma spp. on disease and growth and dry weight of Vigna unguiculata
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Soil inoculation with M. phaseolina significantly decreased length and biomass by 48 and 63%, respectively, with respect to the negative control. There was ~ 55, 100, and 150% increase in said growth attributes of cowpea due to soil amendment with leaf biomass (1–3%). In MP + T. harzianum, length and dry biomass were significantly improved by 159 and 227%, respectively, in combination with leaf biomass of A. indica (1–3%) by 200 and 450% over positive control. When T. viride was provided alone or combined with 1–3% leaf biomass of A. indica, the studied parameter was considerably enhanced by 117–255% over positive control (Table 3).

Highest disease incidence and the maximum reduction in cowpea plant growth attributes due to M. phaseolina inoculation might be ascribed to effect of fungal toxins that could hinder uptake of important minerals in plants, thus disturb the normal functioning of plant possibly by increasing respiration rate, membrane degradation, abnormal stomatal behavior, and abrupt transpiration with excessive loss of water (Heiser et al. 1998).

All biofungicides effectively managed disease by improving growth and physiological attributes in cowpea plants. Soil incorporation with T. harzianum proved more effective as compared to T. viride and dry leaf biomass of A. indica. In either case, combined application of Trichoderma spp. with leaf biomass showed a better effect on cowpea as compared to either biofungicides given alone. However, T. harzianum in combination with dry leaf biomass of A. indica showed the maximum disease management and improvement in plant growth. Lower level of disease incidence and improvement in plant growth attributes after incorporation of soil amendments could be due to their antifungal action that could be further ascribed to enhancement in host resistance, induction of a hypersensitive response through inhibiting growth of M. phaseolina, and conservation of root system function (Vinale et al. 2008). As the Trichoderma species exhibit the ability to grow fast and produce large spore that would be another factor behind the disease suppression as Trichoderma can uptake nutrients more efficiently as compared to a pathogen (Vinale et al. 2008). Besides improvement in soil texture, soil physicochemical properties with better aeration may provide a more suitable environment for the beneficial microbes as compared to the pathogen. The net results of soil amendments seemed to improve plant physiology ultimately resulting in better plant health. Likewise, allelochemicals effect induced by leaves biomass of A. indica might have antagonist effect on the pathogen. Under combined effect of T. harzianum and leaves biomass of A. indica, it appears that disease causing ability of the pathogen have been shifted towards its survival under stress conditions imposed by fungicidal action of allelochemicals and competition for resources between the pathogen and antagonistic fungi.

Effect on plant physiology

Effect on total chlorophyll content

Total chlorophyll content was significantly declined by 60% over negative control due effect of pathogen. Application of different doses of dry leaf manure (1–3%) significantly increased the said attribute up to 165–195% over positive control. In MP + T. harzianum, this parameter was improved by 230% and more profoundly by 265–320% in MP + T. harzianum + A. indica (1–3%). Effect of MP + T. viride and MP + T. viride + A. indica (1–3%) was also significant in improving total chlorophyll content by 200 and 220–255%, respectively, over positive control (Table 4). Damage to thylakoid membrane, reduction of the ribulose 1-5 biphosphate regeneration and overall disturbance in plant photosynthesis network (Petit et al. 2006) could be a cause of decline in total chlorophyll content of cowpea leaves after pathogen infection. Fungicidal action of leaf biomass of plant and Trichoderma spp. enhanced plant physiology that may direct synthesis of chloroplast enzymes due to which rubisco activity was enhanced (Khodary 2004) resulted in an increase of the total chlorophyll content.

Table 4 Effect of Macrophomina phaseolina, soil amendment, and Trichoderma spp. on physiological attributes in Vigna unguiculata
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Effect on enzyme activities

CAT, POX, PPO, and PAL activities were significantly enhanced ~ two-folds due to effect of M. phaseolina over negative control and incorporation of various biofungicides significantly reduced it over positive control. The highest reduction of 30–50% in enzymes activities were recorded in MP + T. harzianum and in combination with leaves dry biomass over positive control. Likewise, MP + A. indica (1–3%) and MP + T. viride or MP + T. viride + A. indica (1–3%) showed significant reductions of 20–30% in enzymes activities over positive control (Table 4). Generation of reactive oxygen species (ROS), such as superoxide anion and hydrogen peroxide during so-called “oxidative burst,” are the earliest responses, following successful pathogen recognition. ROS may be directly involved in pathogen killing, strengthening of plant cell walls, triggering hypersensitive cell death and systemic resistance signaling (Shetty et al, 2007). An increase in the investigated physiological traits revealed that biochemical defense responses shown by cowpea were a reaction of damage caused by M. phaseolina, but not as an efficient defense mechanism resulting compatible host-pathogen interaction in positive control. Soil amendments markedly decreased levels of antioxidant enzymes that could be related to the fact that when antagonistic fungi and allelopathic plant leaf biomass were applied, these agents normalized the effects so cowpea plant would have to face in case of pathogen attack.

Conclusions

It was concluded that application of T. harzianum in combination with leaf biomass of A. indica was effective and environmentally friendly method of managing charcoal rot of cowpea. Thus, reduction in disease incidence and improvement in plant growth through altering host plant physiology resulted in increasing resistance in the cowpea plant through suppression of ROS scavenging enzymes against charcoal rot disease.