Effect of Application Time on the Efficacy of <i>Trichoderma</i> spp. to Biologically Control Sunflower Charcoal Rot

Macrophomina phaseolina (Tassi) Goid, causing charcoal rot disease of soybean, is one of the major factors threatening soybean production, especially in dry years. This pathogen remains the prevailing causal agent of charcoal rot disease that significantly suppresses the yield of a variety of oilseed crops. Its wide host range and ability to survive under arid conditions, coupled with the ineffective use of fungicides against it, have spurred scientific endeavours for alternative avenues to control this phytopathogen. Hence, the present study aimed to provide empirical evidence of the efficacy of fungal isolates of Trichoderma spp. as biological control agents against charcoal rot in soybean (Glycine max L.). In this study Trichoderma harzianum strains 6, 14, 17, 21, 44, T. asperellum 26 and T. virens 32 were evaluated as potential biological agents for control of this disease. Mycelial growth of M. phaseolina strain h-7 was reduced by cell-free and volatile metabolites of Trichoderma strains by 16.4 to 64.8%. T. harzianum strain Tj17 significantly (p≤0.05) reduced the incidence (to 7.3%) and severity (to 3%) of disease 42 days after inoculation and increased the 1000 grain weight (to 178 g) in greenhouse conditions. For confirmation of the greenhouse tests, the selected antagonists were re-examined in field trials, where this isolate reduced the disease incidence (to 10%) and severity (to 3%). The overall results of this study show high capability of used antagonists in reduction of disease severity and incidence, and resulting in increased weight of the product. Hence, the findings reported in the present study supported the applicability of Tj17 isolate as possible alternative to fungicides for the control of charcoal rot in soybean.

harcoal rot (Macrophomina phaseolina (Tassi) Goidanich) of soybean (Glycine max (L.) Merr.) is a disease of economic significance throughout the world.Pathogenicity of 14 isolates of Macrophomina phaseolina was tested on soybean cv.Giza 21 under greenhouse conditions.They capable to infect soybean plants caused charcoal rot on the basil stem with various degrees of diseases severity.Isolate S13 caused the highest charcoal rot severity (60%) followed by isolates S11 and S8 (57.9 and 56.3 %, respectively).The positive effect of two inducer chemicals, i.e riboflavin (vitamin B2) and.thiamine (vitamin B1) on the induction of systemic resistance in soybean against charcoal rot disease as well as biochemical changes associated with these treatments in soybean plants was recorded.Under greenhouse conditions, the dose effect of 0.1 to 15 mM riboflavin and thiamine showed that 2.5 mM of riboflavin and 5 mM of thiamine was sufficient for maximum induction of resistance; higher concentration did not increase the effect.On the other hand, plants treated with riboflavin and thiamine and inoculated with pathogen grow higher than plants treated with sterilized distilled water (SDW) and inoculated with pathogen, while increased fresh and dry weight of soybean plants.At 10 mM concentration of riboflavin and thiamin recorded the highest dry and fresh weights.In time course observation, it was observed that riboflavin and thiamine treated soybean plants were inducing resistance one day after treatment and reached its maximum level from 5 to 7 days in case of riboflavin and 6-8 days in case of thiamine and then decreased.Under field conditions, the percentages of damping-off, root rot and/or charcoal rot severity were significantly reduced due to soaking the seeds in any of riboflavin and thiamine before sowing in both trial seasons (2008-2009 and 2009-2010).Also, these treatments significantly increased nodule numbers plant -1 , fresh and dry weight of nodules plant -1 in both experimental seasons.Generally, thiamine gave the best results in most cases under greenhouse and field conditions.In physiological studies, activity of defense-related enzymes, including peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, pathogenesis related (PR) protein (chitinase), were increased in inoculated and non-inoculated plants treated with the thiamine and riboflavin individually, during the experimental period.In general, activity of these enzymes begins to accumulate after two days of treatment and reached maximum levels at 8, 6, 8 and 8 days for PO, PPO, PAL and chitanase, respectively, then the activities of these C

  Charcoal rots (Macrophomina phaseolina (Tassi) Goidanich) of soybean (Glycine max (L.) Merr.) is a disease of economic significance throughout the world. Pathogenicity of 14 isolates of M. phaseolina was tested on soybean cv. Giza 21 under greenhouse conditions. The obtained data indicated that all the obtained isolates were able to attack soybean plants and caused charcoal rot on the basal stem with various degrees of diseases severity. M. phaseolina isolate S13 caused the highest charcoal rot severity (60%) followed by isolates S11 and S8 (57.9 and 56.3%, respectively). The effect of two inducer chemicals, that is, riboflavin (B2) and thiamine (B1) on the induction of systemic resistance in soybean against charcoal rot disease as well as biochemical changes associated with these treatments in soybean plants was investigated. Under greenhouse condition, the dose effect of 0.1 to 15 mM riboflavin and thiamine showed that 2.5 mM of riboflavin and 5 mM of thiamine was sufficient for maximum induction of resistance; higher concentration did not increase the effect. On the other hand, plants treated with riboflavin and thiamine and inoculated with pathogen grew higher than plants treated with sterilized distilled water (SDW) and inoculated with pathogen and increased fresh and dry weight of soybean plants. 10 mM concentration of riboflavin and thiamin recorded the highest dry and fresh weight compared with the control. In time course observation, it was observed that riboflavin and thiamine treated soybean plants induced resistance one day after treatment and reached its maximum level from 5 to 7 days in the case of riboflavin and 6 to 8 days in the case of thiamine and then decreased. Under field conditions, the percentage of damping-off, root rot and/or charcoal rot severity were significantly reduced due to soaking of the seeds in any of riboflavin and thiamine before sowing compared with the control treatment (seed treated with water) in both seasons (2008 to 2009 and 2009 to 2010). Also, these treatments significantly increased nodule numbers per plant, fresh and dry weight of nodules per plant compared with the control in both seasons. Generally, thiamine gave the best results in most cases under greenhouse and field conditions. In physiological studies, activity of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), pathogenesis related (PR) protein (chitinase), were increased in the inoculated and non-inoculated plants treated with the thiamine and riboflavin respectively, compared with the control during the experimental period. In general, activity of these enzymes began to accumulate after two days of treatment and reached maximum levels at 8, 6, 8 and 8 days for PO, PPO, PAL and chitinase, respectively then the activities of these enzymes decreased progressively. On the other hand, total phenols and lignin increased in soybean plants inoculated with M. phaseolina and treated with thiamine and riboflavin. The highest accumulation of phenols was recorded 6 days after treatment, while lignin recorded the highest level at the 10th day from application. These results suggested that these chemicals mayplay an important role in controlling the soybean charcoal rot disease, through induction of systemic resistance in soybean plants.   Key words: Soybean, charcoal rot, thiamine, riboflavin, induced resistance.

Sunflower (Helianthus annuus L.), a member of the Asteraceae, is one of the major oilseed crops around the world. Charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is the most damaging disease of sunflowers globally. Fungicides are mostly used to control charcoal rot; however, these cause environmental pollution and pose adverse effects on the ecosystem. Therefore, ecofriendly management options are inevitable for the management of charcoal rot disease. Plant mineral nutrition, the use of plant growth-promoting rhizobacteria and biochar have recently been manipulated for the management of different plant diseases. However, the interactive effects of all these treatments have rarely been tested on charcoal rot suppression in sunflowers. This study assessed the influence of sulfur (0 and 2.25 mg/kg) combined with farmyard manure biochar (2%), NPK (20:20:20 mg/kg) and three different plant growth-promoting rhizobacteria (PGPR) strains on the charcoal rot suppression growth, yield, biochemistry and physiology of sunflower. The PGPR strains included in the study were Bacillus sp. strain MR-1/2 (regarded as PGPR1), Achromobacter sp. strain FB-14 (regarded as PGPR2) and Planomicrobium sp. strain MSSA-10 (regarded as PGPR3). The charcoal rot infestation was induced by inoculating the soil with M. phaseolina, and the impacts of the different treatments were studied on the disease infestation, growth, yield, biochemistry and physiology of sunflowers under 0 and 2.25-mg/kg S application. The results revealed that farmyard manure biochar and Planomicrobium sp. strain MSSA-10 in combination with 2.25-mg/kg S proved effective for the management of charcoal rot disease through regulating the antioxidant enzymes’ activities and strengthening the immune system of sunflower plants. The studied health markers (total chlorophyll content and carotenoids) and stress markers (total protein content, catalase and peroxidase) were significantly altered by the applied treatments under 0 and 2.25-mg/kg S applications. The findings of the experiment indicated that both farmyard manure biochar and Planomicrobium sp. strain MSSA-10, combined with 2.25-mg/kg S, could be used to enhance the crop yield and manage charcoal rot disease in sunflowers. Farmyard manure biochar and Planomicrobium sp. strain MSSA-10 are an easy-to-apply, cost-effective, ecofriendly and sustainable option for the management of charcoal rot disease in sunflowers.

In our earlier investigation, a fungal isolate Penicillium citrinum VFI-51 and its secondary metabolite was reported to have antagonistic potential against Botrytis cinerea, the causative agent of Botrytis gray mold disease in chickpea. In the present investigation, P. citrinum VFI-51 was further evaluated for its antagonistic potential against Macrophomina phaseolina, the causative agent of charcoal rot in sorghum. P. citrinum VFI-51 inhibited M. phaseolina in both dual culture as well as secondary metabolite production assays. In the in vivo blotter paper assay, under light chamber conditions, P. citrinum VFI-51 controlled 85% of the charcoal rot disease on the roots when compared to the positive control. Under greenhouse conditions, when M. phaseolina was inoculated by tooth pick method in to the stalk of sorghum plant, the charcoal rot disease was controlled by 75% in P. citrinum VFI-51 treatment over the positive control. This study demonstrates the biocontrol potential of P. citrinum VFI-51 against charcoal rot of sorghum.   Key words: Macrophomina phaseolina, charcoal rot, sorghum, Penicillium citrinum VFI-51, biocontrol.

Macrophomina is a genus belonging to Botryosphaeriaceae that comprises well-known necrotrophic pathogens related to hundreds of plant hosts around the world. Historically, M. phaseolina is the causal agent of charcoal rot in several crops, mainly in tropical and subtropical areas around the world. However, after a recent genetic diversity study using morphological and molecular approaches, which resulted in the epitypification of M. phaseolina, and the description of a new Macrophomina species associated with charcoal rot disease, the hypothesis that other cryptic species could be present under the name M. phaseolina was raised. Previous studies in Brazil revealed a high genetic diversity and different levels of aggressiveness of M. phaseolina isolates associated with charcoal rot in oilseed crops. Thus, the aim of the present study was, through phylogenetic and morphological studies, to determine if isolates of Macrophomina obtained from different oilseed crops represent a single species or distinct taxa. Based on the results obtained, it was possible to identify three different Macrophomina species: M. phaseolina, M. pseudophaseolina and a new phylogenetic species, M. euphorbiicola. This is first report of M. pseudophaseolina in Brazil causing charcoal rot on Arachis hypogaea, Gossypium hirsutum and Ricinus communis and associated with seed decay of Jatropha curcas. In addition, a novel species described in the present study, M. euphorbiicola, is reported as the etiological agent of the charcoal rot on R. communis and Jatropha gossypifolia.

Charcoal rot disease, a root and stem disease caused by the soil-borne fungus Macrophomina phaseolina (Tassi) Goid., is a major biotic stress that limits sorghum productivity worldwide. Charcoal rot resistance-related parameters, e.g., pre-emergence damping-off%, post-emergence damping-off%, charcoal rot disease severity, and plant survival rates, were measured in a structured sorghum population consisting of 107 landraces. Analysis of variance of charcoal rot resistance-related parameters revealed significant variations in the response to M. phaseolina infection within evaluated accessions. Continuous phenotypic variations for resistance-related parameters were observed indicating a quantitative inheritance of resistance. The population was genotyped using 181 simple sequence repeat (SSR) markers. Association analysis identified 13 markers significantly associated with quantitative trait genes (QTLs) conferring resistance to charcoal rot disease with an R2 value ranging between 9.47 to 18.87%, nine of which are environment-specific loci. Several QTL-linked markers are significantly associated with more than one resistance-related parameter, suggesting that those QTLs might contain genes involved in the plant defense response. In silico analysis of four novel major QTLs identified 11 putative gene homologs that could be considered as candidate genes for resistance against charcoal rot disease. Cluster analysis using the genotypic data of 181 SSR markers from 107 sorghum accessions identified 12 main clusters. The results provide a basis for further functional characterization of charcoal rot disease resistance or defense genes in sorghum and for further dissection of their molecular mechanisms.

Streptomycesalbus strain CAI-21 has been previously reported to have plant growth-promotion abilities in chickpea, pigeonpea, rice, and sorghum. The strain CAI-21 and its secondary metabolite were evaluated for their biocontrol potential against charcoal rot disease in sorghum caused by Macrophomina phaseolina. Results exhibited that CAI-21 significantly inhibited the growth of the pathogen, M. phaseolina, in dual-culture (15 mm; zone of inhibition), metabolite production (74% inhibition), and blotter paper (90% inhibition) assays. When CAI-21 was tested for its biocontrol potential under greenhouse and field conditions following inoculation of M. phaseolina by toothpick method, it significantly reduced the number of internodes infected (75% and 45% less, respectively) and length of infection (75% and 51% less, respectively) over the positive control (only M. phaseolina inoculated) plants. Under greenhouse conditions, scanning electron microscopic analysis showed that the phloem and xylem tissues of the CAI-21-treated shoot samples were intact compared to those of the diseased stem samples. The culture filtrate of the CAI-21 was purified by various chromatographic techniques, and the active compound was identified as “organophosphate” by NMR and MS. The efficacy of organophosphate was found to inhibit the growth of M. phaseolina in the poisoned food technique. This study indicates that S. albus CAI-21 and its active metabolite organophosphate have the potential to control charcoal rot in sorghum.

A field trial was conducted at Mallawi Agricultural Research Station, Minia Governorate, ARC, during 2016 and 2017 seasons in a high naturally infested field with charcoal rot and wilt diseases complex, as well as under artificially infestation with charcoal rot and wilt pathogens i.e. Macrophomina phaseolina and Fusarium oxysporum, respectively. The aim of this working to study the effect of intercropping guar with sesame at three planting densities of 100, 67 and 50% from sole guar and number of once and twice to reduce charcoal rot and wilt diseases complex, and yield and yield components of both crops. All intercropping patterns were significantly decreased disease incidence of charcoal rot and wilt diseases compared to the sole sesame under field and pots conditions. The intercropping 100% Sesame + 100% guar was the best pattern to reduce the incidence of charcoal rot & wilt disease complex in both seasons and pots experiment. Sesame seed yield/fad with guar at 50% plant density was increased by 32.66, 23.27% and 28.51, 18.58% than those of guar at of 100% and 67% in the 1 st and 2 nd, seasons, respectively. The highest value of total fresh yield/fad (ton) and total dry yield/fad (ton) of guar with sesame at intercropping pattern 100% guar were 5.91, 0.79 and 6.69, 0.69 ton/fad in the first and second seasons, respectively. Intercropping 50% guar with sesame and one cut guar recorded the highest values for LER, net return and MAI (monetary advantage index) in both seasons. Meanwhile, the lowest net return was recorded for intercropping 100% with two cuts of guar. Our study concluded that intercropping crop guar can be used for the protection of sesame charcoal- rot and wilt disease complex and produced greater seed yield than either crops grown alone.

Two field experiments were carried out at 6 October farm, El-Nubaria Province, the desert backyard of El-Behaira Governorate- Egypt during the two successive summer seasons (2010 and 2011) to study the effect of the integration between different sowing methods and different bio fertilization treatments on sunflower (Helianthus annuus L.) var. Giza-102 productivity and charcoal rot disease management caused by Macrophomina phaseolina (Tassi) Goidunder the reclaimed soil conditions. Rides sowing methods were found to be more effective in reducing M.phaseolina population and charcoal rot, therefore enhance sunflower productivity compared to the other examined sowing methods. EM-X { EM1 + Bacillus subtilis + mycorrhiza + Azotobacter sp.} was found to be more effective in reducing M.phaseolina population and charcoal rot, therefore enhance sunflower productivity compared to the other examined biofertilization treatments including the conventional chemical fertilization as the control treatment. The integration between ridges sowing method with tillage and EM-X{ EM1 + B. subtilis + mycorrhiza + Azotobacter sp.} was found to be the furthermost advised agricultural practices under these conditions as land degradation neutrality (LDN) technology that enhances land cover and area unit productivity under these conditions.

Background: Charcoal rot disease, caused by the soil-borne pathogen Macrophomina phaseolina, poses a significant threat to strawberry cultivation in Maharashtra, India. This study highlights critical factors contributing to disease spread, including climate variables, soil characteristics and farming practices. We propose a comprehensive set of recommendations for improved disease management, aiming to enhance strawberry production and economic outcomes for farmers in Maharashtra. Methods: This study presents an extensive survey of charcoal rot incidence, severity and management practices in the region. Through rigorous field surveys conducted across 100 strawberry farms during 2020 - 2022 laboratory analyses of 2000 plant samples and detailed interviews with 300 local farmers, we identified the prevalence of the disease, its impact on yield and quality and the effectiveness of current management strategies. Result: Our findings reveal that 47% of surveyed farms reported charcoal rot incidence, with an average Disease Severity Index of 3.4 on a 5-point scale. Affected farms experienced yield reductions of 18-35% and significant decreases in fruit quality, with economic losses estimated at 150,000-400,000 per hectare. Chemical treatments and crop rotation are commonly employed, their effectiveness varies considerably. Integrated disease management practices showed the most promise in reducing disease severity, with a 40% reduction in DSI compared to conventional methods.

Biosynthesized silver nanoparticles using Trichoderma harzianum reduce charcoal rot disease in bean

Sesame (Sesamum indicum L.) is one of the most important oilseed crops in Egypt and worldwide. It is being infected with many pathogens, among these pathogens Fusarium oxysporum f.sp. sesami (Zap.) Cast is causing severe economic losses on sesame. In this study, antagonistic capability of 24 isolates of Trichoderma spp. was assessed in vitro against F. oxysporum f.sp. sesami. Two strains; T. harzianum (T9) and T. viride (T21) were revealed to have high antagonistic effect against F. oxysporum f.sp. sesami in vitro with inhibition percentage about 70 and 67%, respectively. These two isolates proved to have high ability to control Fusarium wilt disease under greenhouse conditions. The highest reduction in disease severity was achieved with T. viride followed by T. harzianum with reduction in disease severity about 77 and 74%, respectively. This study revealed that the time of application of bioagents is a decisive factor in determining the efficacy of Trichoderma isolates to control Fusarium wilt of sesame. It was revealed that the highest reduction in the disease severity was achieved when either Trichoderma viride or T. harzianum were applied 7 days before challenging with the F. oxysporum f.sp. sesami.

In September 1998, symptoms suggestive of charcoal rot were observed on oilseed sunflower (Helianthus annuus L.) plants in western North and South Dakota. Symptoms first observed on plants approaching physiological maturity consisted of silver-gray lesions girdling the stem at the soil line, premature plant death, and reduced head diameter. The pith in the lower stem was completely absent or compressed into horizontal layers. Black, spherical microsclerotia were observed in the pith of the lower stem, underneath the epidermis, and on the exterior of the taproot. Confirmation of Macrophomina phaseolina (Tossi) Goid. as the causal agent was based on the size of the microsclerotia, which ranged from 80 to 90 μm in diameter, from both infected sunflowers and pure cultures (1). The only other sunflower pathogen known to form microsclerotia is Verticillium dahliae Kleb., whose microsclerotia are irregular in shape and are 15 to 50 μm in diameter. Some prematurely dead sunflower plants lacked typical charcoal rot stem lesions, but contained Macrophomina microsclerotia. Plants with atypical symptoms were colonized by the sunflower stem weevil (Cylindrocopturus adspersus (LeConte)) and the black sunflower stem weevil (Apion occidentale Fall). This agrees with observations in Texas, where Macrophomina-infected sunflower plants parasitized by stem-feeding insects often displayed atypical charcoal rot symptoms (3). Charcoal rot incidence in 1998 in western North Dakota was 25%, compared with 0% in eastern North Dakota. Charcoal rot was not observed in 1999, the fourth wettest growing season on record, but was observed again in 2000 and 2001. The recent increase in sunflower production in western North and South Dakota, areas typically hotter and drier than the eastern portions of both states, and the potential involvement of stem weevils as vectors of Macrophomina (2) may lead to an increased incidence of charcoal rot in sunflower.

This study was carried out in augmented design during rabi 2021 (November‒May) at Agriculture research station, Hagari, Karnataka, India to identify the charcoal rot resistant mutant line. Total 200 mutants and 7 checks were used to study the charcoal rot resistance in the present experiment. Charcoal rot is a major disease in the dry sorghum-growing regions of Asia, Africa, Americas and Australia. Charcoal rot disease is caused by Macrophomina phaseolina (Tassi) Goid. It appears in severe form on the improved varieties in hot dry weather with soil moisture stress. The process of mutation is recognized as one of the driving forces of evolution. Induced mutation breeding is a relatively quick method of creating variability in quantitatively inherited traits between plants. The parameters used in charcoal rot studies were lodging per cent, mean number of nodes crossed, mean length of spread and Charcoal rot index (CRI). The screening results revealed that 66 mutant lines shown moderate resistant reaction compared to the resistant check DSV-4 (0.5) and E-36-1 (0.27), among them eight mutants had exact only one node crossed by the pathogen. These mutant lines exhibited comparatively lowest number of mean nodes crossed. 84 mutant lines shown moderate resistant response to charcoal rot index trait. These resistant lines can be used for further confirmation and also for future resistant breeding programme.