Clay-based azoxystrobin formulation enhances cotton protection against charcoal rot disease

The fungus Macrophomina phaseolina causes charcoal rot disease (CRD) in cotton, whose symptoms develop in the late stages of growth and result in wilting and death. Despite significant research efforts to reduce disease incidences, effective control strategies against M. phaseolina are an ongoing scientific effort. Today's CRD control tends toward green options to reduce the chemicals' environmental footprint and health risks. Here, different Trichoderma species were examined separately and in combination with Azoxystrobin (AS) in semi-field open-enclosure pots and a commercial field throughout a full season. In the pot experiment, the T. asperellum (P1) excelled and led to improvement in growth (13%-14%, day 69) and crops (the number of capsules by 36% and their weight by 78%, day 173). The chemical treatment alone at a low dose had no significant impact. Still, adding AS improved the effect of T. longibrachiatum (T7507) and impaired P1 efficiency. Real-time PCR monitoring of the pathogen DNA in the plants' roots at the harvest (day 176), revealed the efficiency of the combined treatments: T. longibrachiatum (T7407 and T7507) + AS. In a commercial field, seed dressing with a mixture of Trichoderma species (mix of P1, T7407, and Trichoderma sp. O.Y. 7107 isolate) and irrigation of their secreted metabolites during seeding resulted in the highest yields compared with the control. Applying only AS irrigation at a low dose (2,000 cc/ha), with the sowing, was the second best in promoting crops. The molecular M. phaseolina detection showed that the AS at a high dose (4,000 cc/ha) and the biological mix treatments were the most effective. Reducing the AS chemical treatment dosages by half impaired its effectiveness. Irrigation timing, also studied here, is proven vital. Early water opening during the late spring suppresses the disease outburst and damages. The results demonstrated the benefits of CRD bio-shielding and encouraged to explore the potential of a combined bio-chemo pest control approach. Such interphase can be environmentally friendly (reducing chemical substances), stabilize the biological treatment in changing environmental conditions, achieve high efficiency even in severe CRD cases, and reduce the development of fungicide resistance.

ABSTRACTThe present study was carried out to optimize the physical and nutritional parameters for cultivation of Trichoderma harzianum Rifai for production of extracellular β-glucosidase, an alternative to chemcial fungicides to control Macrophomina phaseolina, which causes charcoal rot disease in crops. Response surface methodology by the Box–Behnken design (BBD) based on four factors (temperature, carbon sources, inoculum size and pH) was used for optimization of the cultivation conditions to produce β-glucosidase using agro-industrial banana wastes. The highest β-glucosidase activity (1483.27 U/mL) was attained under optimized cultivation conditions: ∼36 ºC and pH 7.3, using carbon source and inoculum size of 10% (w/v) and 5% (w/v), respectively. It is noteworthy that the low deviation values (0.09−0.44%) in the verification experiments (R1, R2 and R3) inferred the generated model was accurate to predict optimal cultivation conditions of T. harzianum Rifai. Likewise, the obtained diameters of inhibition zones ranging from 58.00−38.66 mm following treatment with β-glucosidase was found comparable to other in vitro tests using pure T. harzianum isolates and chemical fungicides. Hence, the findings indicated that it was feasible to use β-glucosidase as a greener alternative to chemical fungicides for control of M. phaseolina infection and consequently, for protection of crops against charcoal rot disease.

The use of microbial bioinoculants for managing plant diseases and promoting plant growth is an effective alternative approach to integrated farming. One of the devastating phytopathogens is Macrophomina phaseolina (Tassi) Goid. It is an omnipresent fungus infecting more than 500 plant species. It causes charcoal rot disease in soybean leading to 30-50% yield loss. Soybean Glycine max (L.) oil seed crop produced globally is highly susceptible to M. phaseolina. India is the fifth largest producer of soybean in the world. Madhya Pradesh is the largest soybean-producing state in India; Around 70% yield loss of soybean is accounted to M. phaseolina infection in India. Control of charcoal rot is the requisite of the current situation. Chemical control is not feasible due to saprophytic nature and prolonged survival of Macrophomina phaseolina. Chemical fungicides are expensive, toxic, hazardous, and cause pollution. Biological control is an effective approach to control this devastating fungus. The rhizosphere of soil is rich in beneficial microflora competent to suppress plant pathogens and also promote plant growth. PGPR have well-developed mechanisms that impart antagonistic traits to them. PGPR produces various antifungal metabolites siderophores and HCN which inhibit fungal growth, and can be used as potent BCA. Pseudomonas and Bacillus species have been reported effective against M. phaseolina. The mechanisms and antifungal compounds produced by these bacteria to control charcoal rot can be studied extensively. BCA or the metabolites secreted by them have the potential to develop effective bioformulations for soybean at the commercial level for sustainable agriculture.

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, results in major economic losses in soybean production in southern USA. M. phaseolina has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of M. phaseolina isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 µg/mL), 37.8% produced traces below the limit of quantification (0.01 µg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 µg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in M. phaseolina culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, M. phaseolina isolates from soybeans with charcoal rot disease in southern USA.

Controlling agricultural pests using suitable biocontrol agents has been considered the best strategy for sustainable agriculture. Charcoal rot caused by a necrotrophic fungus Macrophomina phaseolina is responsible for a 30–50% annual reduction in soybean yield worldwide. Little is known about the role of Bacillus clausii in reducing charcoal rot disease severity in the soybean crop. In this study, we investigated plant growth promoting and antagonistic potential of Pseudomonas putida (MT604992) and Bacillus clausii (MT604989) against charcoal rot disease incidence in soybean. Among twenty bacteria isolated from soil and water samples of two different hot springs of Gilgit-Baltistan, Pakistan, 80% were siderophore positive; 65% were hydrogen cyanide (HCN) positive; 55%, 30%, and 75% were phosphate, potassium, and zinc solubilizers, respectively. Based on higher antagonistic activities and plant growth promoting traits five strains were selected for in vitro screening. Out of all tested strains, Pseudomonas putida and Bacillus clausii showed a significant increase in germination, growth, and disease suppression in soybean. These strains produced a pronounced increase in relative water content, photosynthetic pigments, membrane stability, proline, antioxidant enzymes status, phytohormones content (Salicylic acid, and Jasmonic acid), and disease suppression in comparison to control plants. Bacillus clausii mitigated the disease by 97% with a marked increase in the proline content (73% and 89%), superoxide dismutase (356% and 208%), peroxidase (439% and 138.6%), catalase (255.8% and 80.8%), and ascorbate peroxidase (228% and 90%) activities in shoots and roots, respectively. Infected plants showed an increase in salicylic acid and jasmonic acid content which was further increased with the application of the selected strains to increase resistance against pathogens. To our knowledge, this is the first study showing a rise in salicylic acid and jasmonic acid in Macrophomina phaseolina infected plants. These two strains are suggested as a cost-effective, eco-friendly, and sustainable alternative to chemical fungicides. However, there is a need to explore the field testing and molecular mechanisms leading to disease suppression by these strains.

Histopathology of charcoal rot disease (Macrophomina phaseolina) in resistant and susceptible cultivars of soybean

Charcoal rot disease of soybean is a major threat to soybean harvests in the United States. This disease, caused by the fungal pathogen Macrophomina phaseolina (MP), cannot be economically treated in agronomic field conditions, and breeding of resistant cultivars has been largely unsuccessful due to multiple gene resistance. For difficult-to-treat phytopathogens, nascent research has emphasized the potential utility of endophyte modulation of disease symptoms. Most research into biocontrol of MP in soybeans has focused on in vitro or limited greenhouse/pot experiments, which may not emulate field conditions. Here, using a paired framework of diseased and asymptomatic soybeans exposed to MP in a production field setting, coupled with model-based community analyses, we identify several fungal endophytic taxa that apparently modulate disease pathogenicity. We use a multilevel analytical framework to provide evidence of taxa that respond to the disease state by querying plants across years and plant compartments. Additional work is needed to test the mechanisms by which these identified taxa may modulate MP, but this work provides the strongest evidence to date that endophytes may modulate charcoal rot disease levels in production field conditions. We posit that breeding for improved colonization of disease-modulating endophytes is a promising option to manage charcoal rot in soybean. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Objective Melon charcoal rot is one of the most important diseases that significantly reduces the yield of melon (Cucumis melo L.). The main objectives of this study were: isolation of actinomycetes from the rhizospheric soil of different melon cultivation regions located in the Kerman and Sistan and Baluchestan provinces of Iran; investigating the antagonistic effects of actinomycetes against melon black stem or charcoal rot disease; evaluation of siderophore production by isolates in vitro; identification of potential isolate by PCR, and investigating their biocontrol efficacy against Macrophomina phaseolina in melon under greenhouse condition. Materials and methods Eighty actinomycete isolates were isolated from the soil of selected different melon cultivation areas and their antifungal activity against Macrophomina phaseolina was investigated. Potential isolates were evaluated for biological activities. Moreover, the efficacy of selected actinobacteria in order to biocontrol charcoal rot disease was investigated under greenhouse condition. Results Three actinomycete isolates (R1.6, R5.52 and R5.56) were revealed the highest inhibition zone size against Macrophomina phaseolina and selected for further investigations. All three isolates were able to colonize melon roots, produce extracellular enzymes and control disease in the greenhouse. The actinomycete isolate R5.56 was identified by sequence analysis of small ribosomal RNA subunit (16S rRNA) and based on the results this isolate had the highest similarity (98%) with Streptomyces species. Conclusions Biological control of plant pathogens, unlike the application of chemical pesticides, does not work quickly, but in successful cases, has more long-lasting effects compare to chemical pesticides. Biological control should be mentioned as a key component of the integrated pests and plant diseases management system approaches to minimize the environmental side effects and risks as the consequences of over usage of chemicals. This study is a prelude to further studies such as the use of these antagonists against Macrophomina phaseolina in the field, which must be done for at least three years.

The application of algae has been considered a key element for integrated disease management in sustainable agriculture. These organisms can act as a bio-stimulant for induction of resistance against a variety of abiotic and biotic agents that cause economical loss to crop production globally. Charcoal rot disease caused byMacrophomina phaseolina(Tassi) Goid. is one of the biotic agents restricting strawberry (Fragaria×ananassaDuch.) yield in many cultivation sites. Herein, the foliar application of brown alga (Sargassum angustifolium) was investigated for the reduction of the disease symptoms and improvement of vegetative and reproductive indices in strawberries under greenhouse conditions. The results showed that alga-treated infected plants showed symptom remission. Moreover, vegetative and reproductive indices of alga-treated plants were significantly improved. Biochemical analysis showed that in alga-treated infected plants the total phenol, flavonoids, and total antioxidant activity were significantly increased compared to non-treated infected plants. Furthermore, the content of defense-related enzymes, viz. phenylalanine ammonia-lyase and polyphenol oxidase, were significantly increased in the infected plants pre-treated with the alga extract. Foliar application ofS. angustifoliumextract can induce defense responses in strawberry plants infected byM. phaseolinaleading to improved growth indices of the plants. It can be concluded thatS. angustifoliumextract is a promising source of bio-stimulants for induction of disease resistance against charcoal rot disease in strawberry cultivations.

Charcoal rot disease causes heavy yield losses to many hosts including cowpea (Vigna unguiculata) and groundnut (Arachis hypogea) in Senegal. The causal agent of the disease was a longtime considered to be Macrophomina phaseolina. However, a new Macrophomina species, Macrophomina pseudophaseolina was reported recently to also cause charcoal rot disease alone or in association with M. phaseolina on several hosts in Senegal. Since 1969, charcoal rot has become increasingly important in cowpea and other crops in the Sahel. It was not known if this status is correlated with the occurrence of the new Macrophomina species. This study therefore aimed to investigate the pathogenicity of M. phaseolina and M. pseudophaseolina on three varieties of cowpea under two temperature regimes. Ten M. pseudophaseolina and nine M. phaseolina isolates were tested on three cowpea varieties in a complete randomized design. Plants were grown in infected soil at two growth temperatures (24/34 and 26/36°C) in a climatic chamber. Disease incidence, level of tissue infection and potential primary inoculum produced in cowpea plants were determined 45 days after planting. By and large, the two Macrophomina species showed the same results, except that at 36°C, M. pseudophaseolina induced more disease development than M. phaseolina in the susceptible cv. Mouride. In conclusion, M. pseudophaseolina induces less disease incidence on cowpea at 34°C than at 36°C. At this temperature, it becomes as damageable as M. phaseolina on cowpea. Key words: Macrophomina phaseolina, Macrophomina pseudophaseolina, growth temperature, disease incidence, potential primary inoculum.

Menthol Mint (Mentha arvensis) cultivation is estimated to touch 3,00,000 hectares with an annual production of 30,000 tons of essential oils and export about 75% of the output. In recent years the M. arvensis crop at different locations in northern Indian plains was found severely infected with Macrophomina phaseolina. The M. arvensis cultivars such as Kranti, Shivalik, Himalaya, Gomti, Saksham, Saryu, Kosi, and Kalka developed by CSIR-CIMAP were screened for the resistance source against charcoal rot disease caused by M. phaseolina under glasshouse and field conditions. Our results indicated that M. arvensis cultivars Kranti, Gomati, Saryu were resistant, while Shivalik, Himalaya, Saksham and Kalka were moderately resistant, and Kosi was moderately susceptible. The seven cultivars from eight, showed less significant damage by the M. phaseolina, while Kosi cultivars showed high damages. Host fitness made on the basis of infection fact that one cultivar was non-host; one was the poor host while four cultivars were the good host of M. phaseolina. The studies support that Kranti, Gomati, and Saryu cultivars could be suggestible for sustainable cultivation under IGP region to reduce the M. phaseolina associated diseases and prevent yield losses.

Alternative methods are needed to assess the severity of charcoal rot disease [Macrophomina phaseolina (Tassi) Goid] in soybean [Glycine max (L.)] plant tissue. The objective of this study was to define the relationship between light reflectance properties and microsclerotia content of soybean stem and root tissue. Understanding that relationship could lead to using spectral reflectance data as a tool to assess the severity of charcoal rot disease in soybean plants, thus reducing human bias associated with qualitative analysis of soybean plant tissue and cost and time issues connected with quantitative analysis. Hyperspectral reflectance measurements (400-2490 nm) were obtained with a non-imaging spectroradiometer of non-diseased and charcoal rot diseased ground stem and root tissue samples of six soybean genotypes ("Clark", "LD00-3309", "LG03- 4561-14", "LG03-4561-19", "Saline", and "Y227-1"). Relationships between the reflectance measurements and tissue microsclerotia content were evaluated with Spearman correlation (rs) analysis (p < 0.05). Moderate (rs = ±0.40 to ±0.59), strong (rs = ±0.60 to ±0.79), and very strong (rs = ±0.80 to ±1.00) negative and positive statistically significant (p < 0.05) monotonic relationships were observed between tissue spectral reflectance values and tissue microsclerotia content. Near-infrared and shortwave-infrared wavelengths had the best relationships with microsclerotia content in the ground tissue samples, with consistent results obtained with near-infrared wavelengths in that decreases in near-infrared spectral reflectance values were associated with increases in microsclerotia content in the stem and root tissue of the soybean plants. The findings of this study provided evidence that relationships exist between tissue spectral reflectance and tissue microsclerotia content of soybean plants, supporting spectral reflectance data as a means for assessing variation of microsclerotia content in soybean plants. Future research should focus on the modelling capabilities of the selected wavelengths and on the feasibility of using these wavelengths in machine learning algorithms to differentiate non-diseased from charcoal rot diseased tissue.

Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57–79.9 µg/L), moniliformin (61.8% of cultures at 0.011–12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71–1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012–0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants.

Pseudomonas fluorescens isolate 4-92 induced systemic resistance against charcoal rot disease in chickpea (Cicer arietinum L.) caused by Macrophomina phaseolina (Tassi) Goidanich. Time-course accumulation of pathogenesis-related (PR) proteins (chitinases and glucanases) in chickpea plants inoculated with P. fluorescens was significantly (P = 0.05) higher than in control plants. The level of chitinases and glucanases increased by 6.6- to 7-fold up to 4 days postinoculation; thereafter, little decrease in the activity of PR proteins was observed. Root-colonizing populations of P. fluorescens were at a maximum 2 days after transplantation at different inoculum concentrations, and decreased over time. Inoculation of root tips of chickpea by P. fluorescens, 2,6-dichloroisonicotinic acid, and o-acetylsalicylic acid induced systemic resistance against charcoal rot. Disease was 33 to 55.5% higher in control plants than in plants inoculated with chemical inducers or P. fluorescens. Single treatment of plants with P. fluorescens increased disease resistance by 33%, whereas combined application of P. fluorescens with either of the chemical inducers was most effective in inducing the resistance by 2- to 2.25-fold. The time-course study shows that an interval of at least 2 days was required between induction treatment and challenge inoculation. Biocontrol efficacy of P. fluorescens against charcoal rot disease in chickpea was demonstrated under greenhouse conditions.Key words: biological control, induced resistance, Macrophomina phaseolina, Pseudomonas fluorescens.

Macrophomina phaseolina (Tassi) Goid. is a fungal pathogen causes charcoal rot disease (Sin: Rhizoctonia bataticola) and is responsible for significant yield losses in many plants. In our study, we aimed to evaluate the antagonistic ability of 39 different bacteria, isolated from the fields of sugar beet in 2019, against the pathogen Macrophomina phaseolina isolated from sugar beet, beans and chickpeas. Approximately 31% of the bacteria showed antibiosis effect against the pathogen. It was determined that the effectiveness level of Lelliottia amnigena, Bacillus atrophaeus, B.pumilus and B. cereus (7 isolates) was moderate to high against Macrophomina phaseolina. Bacillus atrophaeus (PTo15-1a) showed the highest efficacy of 80%, 72.94% and 82.35% against Macrophomina phaseolina of chickpea, bean and sugar beet respectively. Lelliottia amnigena (Pto 14-1b) was moderately effective (57.78%) against the chickpea isolate of the pathogen. It was observed that of the seven Bacillus cereus isolates used in the experiment, three isolates (Pto14-1a, Pto12-1b, Pto17-1b) were highly effective against the chickpea pathogen, two (Pto12-1b, Pto14-2b) against bean pathogen, and one (Pto15-1b) against sugar beet isolate. Results have shown varied level of antagonism by different test bacterial against different Macrophomina phaseolina isolates, while the highest level of antibiosis shown by Bacillus atrophaeus against all pathogenic isolates indicated that it can be a potential future bioagent in managing the disease.