Macrophomina phaseolina (Tassi) Goid is a destructive pathogen of cowpea that causes serious charcoal rot disease with significant yield losses. Antifungal activity of three indigenous Ascomycetes viz., Trichoderma harzianum, T. viride, and T. hamatum, and two Meliaceae members, i.e., Melia azedarach L. and Azadirachta indica L. were assessed against the pathogen. Laboratory screening trials with cell-free culture filtrate showed the maximum reduction in growth of M. phaseolina with T. harzianum, followed by T. viride. Various concentrations (1–5%) of methanolic leaf extract of A. indica showed more reduction in fungal biomass than M. azedarach. Pot experiment was performed by T. harzianum, T. viride,and dryleafbiomass of A. indica against M. phaseolina. Results revealed that potted soil amended with T. harzianum in combination with 1–3% dry leaf biomass of A. indica held a significant potential to decrease disease incidence to 20–25% and improve plant growth attributes up to fourfolds over positive control inoculated with M. phaseolina only. Physiology of the host plant was altered due to the incorporation of various soil amendments resulting in reduced activities of antioxidant enzymes (catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase). It was concluded that fungal antagonists and allelopathic chemicals would be an effective and eco-friendly means of managing the charcoal rot disease. Keywords: Allelopathic effect, Antioxidant enzymes, Biological control, Charcoal rot, Plant biomass Background soil-borne necrotrophic fungus Macrophomina phaseo- Cowpea (Vigna unguiculata L.) is one of the most lina (Tassi) Goid. The pathogen is widely distributed in important, oldest, herbaceous legume crop, widely culti- the regions with high temperatures and drought condi- vated for fodder and grain in the Pakistan and semi-arid tions, while it is responsible for infecting more than 500 tropics of the world (Mensack et al. 2010). Its substantial plant species including cowpea, mung bean, chickpea, adaptation to drought, elevated temperatures, a wider sorghum, sunflower, etc. Disease causes wilting of host spectrum of pH, requirement of less fertilizers and min- plant after infection and pathogen keeps on producing imal irrigation relative to many other legumes, increase microsclerotia in senescing shoot tissues which causes its preference by the farmers in improving their further decay of host tissue (Mayek-Perez et al. 2001). socio-economic status and in contributing agricultural There are no effective fungicides or other control productivity. However, cowpea growth and productivity methods to limit M. phaseolina (Gaige et al. 2010). are suppressed by very destructive and economically im- Disease management through utilizing native antagonistic portant charcoal rot disease caused by the seed and soil fungi and allelopathic plants is an attractive alternative among the disease management practices. Trichoderma is a common filamentous biocontrol fungal agent, found al- * Correspondence: firstname.lastname@example.org most in any soil type. The antifungal activity of this genus Institute of Agricultural Sciences, Punjab University, Lahore, Pakistan © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Shoaib et al. Egyptian Journal of Biological Pest Control (2018) 28:26 Page 2 of 7 is associated with improvement in growth and systemic 12 days. Extracts were obtained from soaking materials resistance in plant (Harman et al. 2006). Several antagon- by filtering and evaporating and finally drying. Original istic mechanisms like nutrient competition, antibiotic concentration was made by dissolving 9 g of extracting production, and mycoparasitism generally work in Tricho- plant material in 5 ml of dimethyl sulphoxide (DMSO derma against the pathogen (Vinale et al. 2008). Many 99.5%) to prepare a final volume of 15 ml. Control solu- biocontrol mycoparasitic species of Trichoderma have tion was made by adding 5 ml of DMSO in 10 ml of been well studied including T. harzianum, T. viride, T. sterilized distilled water. Six concentrations, i.e., 0, 1, 2, hamatum, T. koningii,and T. reesei against M. phaseolina 3, 4, and 5%, were made by adding 0, 1, 2, 3, 4, and 5 ml (Khalili et al. 2012), and many have been developed into a of stock solution and 5, 4, 3, 2, 1, and 0 ml of control commercial biocontrol product. solution in 55 ml of each flask to make a final volume of Utilization of plant extract and biomass is another envir- medium 60 ml. Then, 60 ml of each treatment was onment friendly way of managing the disease as a source equally divided into four 100-ml flasks to serve as repli- of natural pesticides. Plants are store house of biochemi- cates, where 0% was control treatment. Actively growing cals that contribute in suppressing phytopathogens (Sales culture of M. phaseolina (5 mm disc) was inoculated in et al. 2016). These biochemicals (nitrogen-containing each flask and incubated at 28 ± 2 °C for 7 days. The compounds and phenolics) function as a defense and fungal biomass was dried and weighed. chemical signal molecule against pathogens. Previous literature showed that phytochemicals of Melia azedarach Pot bioassays and Azadirachta indica, besides holding medicinal values, On the basis of the laboratory bioassays, two species of have shown considerable fungicidal activity against patho- Trichoderma viz. T. harzianum and T. viride, and one genic fungi including M. phaseolina (Carpinella et al. member of Meliacaceae family, i.e., A. indica,were 2003). The present study was planned to investigate selected to conduct trials in pots (6 in. diameter × 10 in. −1 antifungal activity of three indigenous Ascomycetes fungal height). Initially, presterilized potted soil (1 kg pot ) was species viz., T. harzianum, T. viride,and T. hamatum,and inoculated with cultural suspension (conidial count 4 × two Meliaceae members, i.e., M. azedarach and A. indica 10 )ofeach of two Trichoderma spp. and left for 4 days against M. phaseolina responsible for charcoal rot disease for the establishment of the fungus in soil. Later dry leaves in cowpea through in vitro trials. 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 Materials and methods (MP) and left for another 4 days for inoculum establish- Laboratory bioassays ment. Finally, surface sterilized seeds of cowpea with 0.1% Three Trichoderma species, i.e., T. viride (FCBP 644), T. sodium hypochlorite solution were sown in each pot. The harzianum (FCBP 1277), and T. hamatum (FCBP 907), pots were arranged in a completely randomized design were tested for their antagonism activity against M. pha- and were kept under natural environmental conditions seolina (FCBP 0751). having three replicates of each treatment. Experiment was comprised of 13 treatments including T :negativecontrol Antifungal activity of Trichoderma spp. by cell-free culture (without any inoculation or amendment); T :positivecon- filtrates trol (inoculated with MP only); T –T :MP+1% A. indica, 3 5 Cell-free culture filtrates Trichoderma spp. were pre- MP + 2% A. indica and MP + 3% A. indica; T :MP+ T. pared in 2% ME (malt extract) broth medium (100 mL). harzianum; T –T :MP+ T. harzianum +1% A. indica, 7 9 After 20 days of inoculation, cell-free supernatants were MP + T. harzianum +2% A. indica,MP+ T. harzianum + collected after aseptic filtration through Whatman filter 3% A. indica; T :MP+ T. viride; T –T :MP+ T. viride 10 11 13 paper and centrifugation at 4000 rpm for 5 min, +1% A. indica;MP+ T. viride +2% A. indica, and MP + followed by re-filtration through Millipore filter paper T. viride +3% A. indica. (pore size 45 μm). Twenty-one different concentrations, ranging from 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, Disease assessment 60, 65, 70,…, 100% (v/v) of each cell-free culture filtrate, After 40 days of inoculation charcoal rot disease symp- were prepared by addition of 2% of ME. Flasks were in- toms on cowpea plants, were appeared and were oculated with 5 mm disc of M. phaseolina and incubated assessed, using disease rating scale, where 1: no symp- at 28 ± 2 °C. After 7 days, mycelial mat was dried in oven toms on plants (highly resistant); 3: lesions are limited to at 45 °C for 24 h for measuring dry biomass. cotyledonary tissues (resistant); 5: lesions have progressed from cotyledons to about 2 cm of stem tis- Assessment of the antifungal potential of plant extract sues (tolerant); 7: lesions are extensive on stem and Two hundred-gram powdered leaves of A. indica and M. branches (susceptible); and 9: most of the stem and azedarach were soaked in 2 L methanol separately for growing points are infected. A considerable amount of Shoaib et al. Egyptian Journal of Biological Pest Control (2018) 28:26 Page 3 of 7 pycnidia and seclerotia are produced (highly susceptible). test (variance test) were applied before any statistical Disease incidence (DI) was determined, using the follow- analysis. When all the assumptions of ANOVA were sat- ing formula: isfied, standard errors of all data were analyzed by analysis of variance (ANOVA), followed by LSD test, Number of infected plants using computer software Statistix 8.1. DIðÞ % ¼ 100 Total nutmber of plants Results and discussion Laboratory trials Analysis of plant physiology Increasing concentrations (5–100%) of cell-free culture Physiological variations in different treatments were filtrate of the three Trichoderma spp. were found highly assessed in cowpea leaves after 40 days of seed sowing. effective in suppressing growth of M. phaseolina. Thus, Total chlorophyll content was quantitatively analyzed by the highest inhibition of 10–90% in the biomass of M. taking absorbance properties for chlorophyll a (645 nm), phaseolina was recorded due to cell-free culture filtrate chlorophyll b (663 nm), and carotenoid (270 nm), and (CFC) of T. harzianum, followed by 5–70% due to CFC the amount of pigment was calculated. Activity of cata- of both T. viride and T. hamatum as compared to con- lase (CAT) was determined in the reaction mixture trol (Table 1). Likewise, Naglot et al. (2015) and Khaledi consisted enzyme extract (0.1 ml) that was added to and Taheri (2016) reported inhibition in growth of M. 2.9 ml of H O (20 mM) and sodium phosphate buffer 2 2 phaseolina by different Trichoderma spp, whereas the (50 mol/L; pH 7.0) by monitoring the reduction in the difference in concentration of volatile substances absorbance at 240 nm (Maehly and Chance 1967). Activ- (acetaldehyde, isocyanide derivatives, terpene, hydrazine, ity of peroxidase (POX) was determined by taking 0.5 ml alcohols, lactones, etc.) and cell wall degrading enzymes of enzyme extract in reaction mixture containing 2 ml of (chitinase and glucanase) might be ascribed to dissimilar 0.1 mol/L phosphate buffer (pH 6.8) and 1 ml of fungicidal activity of three Trichoderma spp. (Woo et al. pyrogallol. Solution was filled with 1 ml of 0.05 mol/L 2006). Considering the significant antifungal activity of H O (5:5 in H O and distilled water), incubated at 25 °C, 2 2 2 2 T. harzianum and T. viride against M. phaseolina, the and reaction was stopped by adding 2.5 mol/L H SO 2 4 two species were later used in the pot study. (24.5 ml of H SO + 100 ml of distilled water). The amount 2 4 In laboratory trials, different concentrations (1–5%) of of purpurogalline formed was determined by reading the leaf extract of M. azedarach and A. indica significantly absorbance at 430 nm against a blank prepared by adding reduced M. phaseolina growth by 19–61 and 25–72%, the extract after the addition of 2.5 mol/L H SO (Colville 2 4 respectively (Table 2). Reduction in fungal biomass to in- and Smirnoff 2008). Polyphenol oxidase activity (PPO) was crease in the concentration of plant extract has been assayed in a reaction mixture consisted of 0.1 ml enzyme reported by several authors (Latha et al. 2009). Many extract and 1.5 ml of 0.1 mol/L sodium phosphate buffer chemicals and biological active compounds have been (pH 7.0), 0.2 ml of 0.01 mol/L catechol. The changes in the identified in the leaf extract of A. indica (phytol, octade- absorbance were recorded at 30-s interval for 3 min at catrienoic acid, methyl ester, hexadecanoic acid, methyl 495 nm (Mayer et al. 1965). For determination of phenyl- ester, etc.) (Hossain et al. 2013) and in M. azedarach alanine ammonia-lyase (PAL) activity, reaction mixture (β-sitosterol, β-amyrin, ursolic acid, benzoic acid, and [(0.4 ml of enzyme extract + 0.1 mol/L sodium borate buf- 3-5 dimethoxy benzoic acid) (Jabeen et al. 2011). The fer (pH 8.8) + 0.5 ml of 0.012 mol/L L-phenylalanine)] was significant reduction in growth of the fungus treated incubated for 1 h in light at 25 °C and reaction was stopped with two plant species was probably due to a difference by incubating at 47 °C for 10 min. The amount of trans- in occurrence of inhibitors to the fungitoxic principle cinnamic acid formed was calculated after measuring (Baka 2010). absorbance of samples at 290 nm (Dickerson et al. 1984). Harvesting and data collection Pot trials Plants were harvested after 65 days of sowing. Data re- Effect on disease and growth garding disease incidence, and plant height, shoot, root There was no disease in negative control. The highest fresh, and dry weight were measured. Materials were disease incidence of 75% was recorded in positive dried at 70 °C, and dry weight was recorded on an control, where only M. phaseolina was inoculated in the electric balance. soil. Soil amendments with 1–3% dry leaf biomass of A. indica significantly reduced the disease incidence from Statistical analysis 50 to 30% over positive control. MP + T. harzianum or Triplicate values reported are mean of ±SD. MP + T. viride significantly reduced disease incidence to Kolmogorov-Smirnov test (normality test) and Levene’s 23 and 39%, respectively. The combined effect of Shoaib et al. Egyptian Journal of Biological Pest Control (2018) 28:26 Page 4 of 7 Table 1 Effect of different concentrations of Trichoderma Table 2 Effect of methanolic extract of Melia azadarch and species filtrate on biomass (g) of Macrophomina phaseolina Azadirachta indica concentrations on biomass (g) of Macrophomina phaseolina Concentrations (%) Trichoderma Trichoderma Trichoderma viride harzianum hamantum Concentration (%) Melia azadarch Azadirachta indica 0 178a 177a 177a 0 166a 355a 5 133b 154b 168ab 1 137b 264b (25%) (13%) (5%) (19%) (25%) 10 118c 153b 160bc 2 128b 218c (33%) (14%) (10%) (24%) (38%) 15 116c 136c 148cd 3 101c 181d (34%) (23%) (16%) (36%) (49%) 20 113cd 125c 143de 4 95d 120e (43%) (29%) (18%) (42%) (66%) 25 100de 124c 132ef 5 64e 97f (44%) (30%) (25%) (61%) (72%) 30 95bef 105d 128f Values with different letters in column show significant difference (P ≤ 0.05) as (46%) (40%) (27%) determined by LSD test. Values represent mean of four replicates and percentage decrease in fungal biomass in parentheses 35 94ef 102d 126f (47%) (42%) (28%) 55, 100, and 150% increase in said growth attributes of 40 89ef 88e 120 fg cowpea due to soil amendment with leaf biomass (49%) (50%) (32%) (1–3%). In MP + T. harzianum, length and dry biomass 45 88ef 83ef 110 g were significantly improved by 159 and 227%, respect- (50%) (53%) (37%) ively, in combination with leaf biomass of A. indica 50 81fg 74fg 92h (60%) (58%) (47%) (1–3%) by 200 and 450% over positive control. When T. viride was provided alone or combined with 1–3% leaf 55 70gh 72fg 90h (61%) (59%) (49%) biomass of A. indica, the studied parameter was considerably enhanced by 117–255% over positive con- 60 69gi 66 g 83hi (63%) (62%) (52%) trol (Table 3). 65 62hj 60 g 73ij Highest disease incidence and the maximum reduction (65%) (65%) (58%) in cowpea plant growth attributes due to M. phaseolina 70 59hj 38h 67jk inoculation might be ascribed to effect of fungal toxins (66%) (78%) (61%) that could hinder uptake of important minerals in 75 45hk 34hi 64jl plants, thus disturb the normal functioning of plant (69%) (80%) (63%) possibly by increasing respiration rate, membrane deg- 80 40il 25hj 62jl radation, abnormal stomatal behavior, and abrupt (71%) (85%) (64%) transpiration with excessive loss of water (Heiser et al. 1998). 85 34jl 22ik 61jl All biofungicides effectively managed disease by im- (71%) (87%) (65%) proving growth and physiological attributes in cowpea 90 30jl 20jk 57kl plants. Soil incorporation with T. harzianum proved (71%) (89%) (67%) more effective as compared to T. viride and dry leaf bio- 95 26kl 17jk 56kl mass of A. indica. In either case, combined application (73%) (91%) (67%) of Trichoderma spp. with leaf biomass showed a better 100 19l 11k 52l effect on cowpea as compared to either biofungicides (74%) (93%) (70%) given alone. However, T. harzianum in combination with Values with different letters in column show significant difference (P ≤ 0.05) as determined by LSD test. Values represent mean of four replicates and percentage dry leaf biomass of A. indica showed the maximum dis- decrease in fungal biomass in parentheses ease management and improvement in plant growth. Lower level of disease incidence and improvement in antagonistic fungi with soil amendment was more pro- plant growth attributes after incorporation of soil nounced. Therefore, T. harzianum with 1–3% dry bio- amendments could be due to their antifungal action that mass of leaf showed the highest reduction in disease could be further ascribed to enhancement in host resist- incidence from 20 to 8% (Table 3). ance, induction of a hypersensitive response through Soil inoculation with M. phaseolina significantly inhibiting growth of M. phaseolina, and conservation of decreased length and biomass by 48 and 63%, respect- root system function (Vinale et al. 2008). As the ively, with respect to the negative control. There was ~ Trichoderma species exhibit the ability to grow fast and Shoaib et al. Egyptian Journal of Biological Pest Control (2018) 28:26 Page 5 of 7 Table 3 Effect of Macrophomina phaseolina, soil amendment, and Trichoderma spp. on disease and growth and dry weight of Vigna unguiculata Treatments Disease incidence (%) Disease severity Height (cm) Biomass (g) T : negative control (without any inoculation or amendment) 131ef 24d–f T : positive control [(inoculated with Macrophomina phaseolina (MP) only)] 75.3a 11a 68g 9h T :MP + 1% A. indica 52.5b 7b 125f 16g T :MP + 2% A. indica 43c 6b 148de 17fg T :MP + 3% A. indica 30.3ef 3c 166cd 23ef T :MP + T. harzianum 22gh 2d 174bc 29cd T :MP + T. harzianum +1% A. indica 18h 2d 191b 41b T :MP + T. harzianum +2% A. indica 11i 1e 213a 45b T :MP + T. harzianum +3% A. indica 6i 1e 223a 52a T :MP + T. viride 38cd 3.5c 147de 23ef T :MP + T. viride +1% A. indica 35de 3.5c 164cd 27c–e T :MP + T. viride +2% A. indica 27fg 2.5d 172bc 28c–e T :MP + T. viride +2% A. indica 21gh 2d 178bc 32c Values with different letters in column show significant difference (P ≤ 0.05) as determined by LSD test. Values represent mean of four replicates produce large spore that would be another factor behind photosynthesis network (Petit et al. 2006)could be acause the disease suppression as Trichoderma can uptake of decline in total chlorophyll content of cowpea leaves nutrients more efficiently as compared to a pathogen after pathogen infection. Fungicidal action of leaf biomass (Vinale et al. 2008). Besides improvement in soil texture, of plant and Trichoderma spp. enhanced plant physiology soil physicochemical properties with better aeration may that may direct synthesis of chloroplast enzymes due to provide a more suitable environment for the beneficial which rubisco activity was enhanced (Khodary 2004)re- microbes as compared to the pathogen. The net results sulted in an increase of the total chlorophyll content. of soil amendments seemed to improve plant physiology ultimately resulting in better plant health. Likewise, alle- lochemicals effect induced by leaves biomass of A. indica Effect on enzyme activities might have antagonist effect on the pathogen. Under CAT, POX, PPO, and PAL activities were significantly combined effect of T. harzianum and leaves biomass of enhanced ~ two-folds due to effect of M. phaseolina over A. indica, it appears that disease causing ability of the negative control and incorporation of various biofungi- pathogen have been shifted towards its survival under cides significantly reduced it over positive control. The stress conditions imposed by fungicidal action of allelo- highest reduction of 30–50% in enzymes activities were chemicals and competition for resources between the recorded in MP + T. harzianum and in combination pathogen and antagonistic fungi. 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 Effect on plant physiology of 20–30% in enzymes activities over positive control Effect on total chlorophyll content (Table 4). Generation of reactive oxygen species (ROS), Total chlorophyll content was significantly declined by such as superoxide anion and hydrogen peroxide during 60% over negative control due effect of pathogen. so-called “oxidative burst,” are the earliest responses, Application of different doses of dry leaf manure (1–3%) following successful pathogen recognition. ROS may be significantly increased the said attribute up to 165–195% directly involved in pathogen killing, strengthening of over positive control. In MP + T. harzianum, this plant cell walls, triggering hypersensitive cell death and parameter was improved by 230% and more profoundly systemic resistance signaling (Shetty et al, 2007). An in- by 265–320% in MP + T. harzianum + A. indica (1–3%). crease in the investigated physiological traits revealed that Effect of MP + T. viride and MP + T. viride + A. indica biochemical defense responses shown by cowpea were a (1–3%) was also significant in improving total chloro- reaction of damage caused by M. phaseolina, but not as phyll content by 200 and 220–255%, respectively, over an efficient defense mechanism resulting compatible host- positive control (Table 4). Damage to thylakoid mem- pathogen interaction in positive control. Soil amendments brane, reduction of the ribulose 1-5 biphosphate markedly decreased levels of antioxidant enzymes that regeneration and overall disturbance in plant could be related to the fact that when antagonistic fungi Shoaib et al. Egyptian Journal of Biological Pest Control (2018) 28:26 Page 6 of 7 Table 4 Effect of Macrophomina phaseolina, soil amendment, and Trichoderma spp. on physiological attributes in Vigna unguiculata Treatments Total chlorophyll CAT U/min per POX U/min per PPO U/min per PAL U/min per content (mg/g) mg of protein mg of protein mg of protein mg of protein T : negative control 0.52e 3.54e 1.43f 0.017cd 0.11c (without any inoculation or amendment) T : positive control 0.19f 5.50a 3.05a 0.04a 0.18a [(inoculated with Macrophomina phaseolina (MP) only)] T :MP + 1% A. indica 0.53e 4.51b 2.41c 0.032b 0.17a (− 165%) (18%) (21%) (20%) (29%) T :MP + 2% A. indica 0.56d 4.12c 2.44c 0.031b 0.15d (− 180%) (25%) (21%) (23%) (29%) T :MP + 3% A. indica 0.59d 4.12c 2.18d 0.031b 0.14b (− 195%) (25%) (29%) (23%) (24%) T :MP + T. harzianum 0.66cd 3.78d 1.80e 0.022c 0.11c (− 225%) (31%) (41%) (45%) (39%) T :MP + T. harzianum +1% A. indica 0.73b 3.73d 1.57f 0.025bc 0.11c (− 265%) (33%) (49%) (38%) (45%) T :MP + T. harzianum +2% A. indica 0.79ab 3.62e 1.44f 0.023c 0.11c (− 295%) (35%) (53%) (43%) (45%) T :MP + T. harzianum +3% A. indica 0.84a 3.61e 1.41f 0.022c 0.09c (− 320) (35%) (54%) (45%) (45%) T :MP + T. viride 0.59d 3.98c 2.58b 0.028bc 0.14b (− 193%) (28%) (15%) (30%) (32%) T :MP + T. viride +1% A. indica 0.64c 3.81d 2.41c 0.029 bc 0.16 ab (−220%) (31%) (21%) (28%) (29%) T :MP + T. viride +2% A. indica 0.67bc 3.71d 2.25d 0.029b 0.15ab (− 235%) (33%) (26%) (28%) (26%) T :MP + T. viride +3% A. indica 0.71b 3.67de 2.16d 0.029b 0.14b (− 255%) (33%) (29%) (27%) (26%) Values with different letters in column show significant difference (P ≤ 0.05) as determined by LSD test. Values in parenthesis show increase/decrease in treatment with respect to positive control and allelopathic plant leaf biomass were applied, these Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in agents normalized the effects so cowpea plant would have published maps and institutional affiliations. to face in case of pathogen attack. Received: 9 November 2017 Accepted: 23 January 2018 Conclusions It was concluded that application of T. harzianum in References combination with leaf biomass of A. indica was effective Baka ZA (2010) Antifungal activity of six Saudi medicinal plant extracts against five phyopathogenic fungi. Arch Phytopathol Plant Prot 43:736–743 and environmentally friendly method of managing Carpinella MC, Giorda LM, Ferrayoli CG, Palacios SM (2003) Antifungal effects of charcoal rot of cowpea. 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Egyptian Journal of Biological Pest Control – Springer Journals
Published: Mar 15, 2018
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