Role of Riboflavin and Thiamine in Induced Resistance Against Charcoal Rot Disease of Soybean

  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.

Rhizoctonia solani was found to be associated with root rot symptoms of guar plants collected from different fields in New Valley governorate, Egypt. All the obtained isolates were able to attack guar plants (cv. Local) causing damping-off and root rot diseases. R. solani isolates No. 8 (RG8) was the more virulent ones in the pathogenicity tests. Salicylic acid (SA) and Pseudomonas fluorescens (PF) individually or in combination were examined for their potential in suppression damping-off and root rot and growth promotion of guar plants in vitro and in vivo. Both SA and P. fluorescens either individually or in combination inhibited the growth of the tested pathogenic fungi.SA combined with P. fluorescens recorded the highest inhibited growth followed by P. fluorescens alone. Under greenhouse and field conditions, all treatments significantly reduced damping-off and root rot severity. The combination of SA and P. fluorescens was more effective than using them individually. Under field conditions, all these treatments significantly increased growth parameters (plant height and No. of branches plant-1) and yield components (No. of pods plant-1, weight of 100 seeds and total yield fed.-1 and Guaran content (gm plant-1) in both locations (El-Kharga and Mallawy Agric. Res. Stations) during growing season 2014.The combination of SA and P. fluorescens were recorded the highest growth parameters and yield components. Generally, the combination of SA and P. fluorescens recorded the best results for controlling damping-off and root rot diseases in greenhouse and field with addition improved plant growth and increased yield components in the field. In physiological studies, activity of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), pathogenesis related (PR) protein (chitinase and β 1,3 gluconase), were increased in inoculated and non-inoculated plants treated with the SA and P. fluorescens either individually or in combination, during the experimental period. The combination of SA and P. fluorescens recorded the highest increase in activity of all enzymes. In general, the activity of these enzymes begins to accumulate after two days of treatment and reached maximum levels at 6 to 10 days, then the activities of these enzymes were decreased progressively. On the other hand, total phenols and lignin increased in guar plants inoculated with R. solani and treated with SA and P. fluorescens individually or in combination. The highest accumulation of phenols was recorded 8th days from application, while lignin recorded the highest level at 10th days from application. In the end, these results suggested that SA and P. fluorescens either individually or in combination may play an important role in controlling the guar damping-off and root rot diseases, though they have induction of systemic resistance in guar plants.

Rhizoctonia solani, Fusarium solani, F. oxysporum F. equiseti and F. semitectum were found to be associated with root rot and wilt symptoms of fodder beet plants collected from different fields in New Valley governorate, Egypt. All the obtained isolates were able to attack fodder beet plants (cv. Starmon) causing damping-off and root rot/wilt diseases. R. solani isolate FB1, F. solani isolate FB7 and F. oxysporum isolate FB11 were the more virulent ones in the pathogenicity tests. The efficacy of 4 different potassium salts for controlling damping-off, root rot and wilt diseases in fodder beet were evaluated in vitro and in vivo. In vitro studies, all the tested potassium salts were significantly suppressed growth of the pathogenic fungi at different concentrations. KHCO3 showed superior higher inhibitory effect on redial growth of the tested pathogenic fungi especially at higher concentration (20 mM). Under green house and field conditions, all potassium salts significantly reduced damping-off and root rot/wilt severity and increased survival of plants. The reduction in damping-off and root rot/wilt increased with increasing of potassium salts concentration except potassium sulfate (K2SO4), while concentration 10 mM was more effective for reducing damping-off and root rot/wilt severity than 20 mM. K2SiO3 followed by K2HPO4 recorded highly protection against damping-off and root rot/wilt severity more than the other tested potassium salts. Under field conditions, all these potassium salts at different concentrations significantly submitted to various growth and yield parameters viz. root length, root diameters, fresh and dry weights compared with control during growing seasons 2013-14 and 2014-15. While, % dry maters was no significant in both growing seasons. The applied treatment K2SiO3 achieved the highest increase in all the mentioned parameters over the other entire three potassium salts during both growing seasons. In physiological studies, activity of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and tyrosine ammonia lyase (TAL) and total phenols content were increased in inoculated plants with R. solani, F. solani, and F. oxysporum individually and treated with potassium salts compared with untreated plants. K2SiO3 at 20 mM showed the highest level of all oxidative enzymes activity and total phenols content followed by K2HPO4 and K2SO4 at 20 mM. Whereas, the least enzymes activity was recorded with KHCO3 at 10 mM. These results suggested that these chemicals may be play an important role in controlling the fodder beet damping-off, root rot and wilt diseases; through they have induction of systemic resistance in fodder beet plants.

This study aimed to assess the role of two Plant growth promoting rhizobacteria (PGPR), Pseudomonas stutzeri (KX574858) and Pseudomonas putida (KX574857) against charcoal rot instigated by Macrophomina phaseolina in soybean (Glycine max L.) varieties; Ajmeri and NARC grown in pots under greenhouse condition. Macrophomina inocula were added to the soil at the time of sowing. Disease incidence and severity were recorded on 90th day of sowing. Seeds were inoculated with PGPR prior to sowing. Growth parameters such as germination index, shoot height and shoot fresh weight were measured at flowering stage. P. stutzeri significantly (p < 0.05) increased germination index (147% and 115%), shoot height (117% and 103%) and shoot fresh weight (120% and 100%) in cv. Ajmeri and cv. NARC, respectively, in infected plants. Both P. stutzeri (76% and 60%) and P. putida (23% and 22%) significantly decreased the disease severity index of charcoal rot in cv. Ajmeri and cv. NARC, respectively. P. stutzeri induced polyphenol oxidase (435% and 386%), phenylalanine ammonia-lyase (257% and 180%), superoxide dismutase (290% and 240%), peroxidase (733% and 666%) and catalase activities (1867% and 1424%) were linearly increased in cv. Ajmeri and cv. NARC, respectively, after 90 days of infection. Significantly higher accumulation of leaf proline and soluble proteins was recorded in both varieties due to P. stutzeri under infected condition. PGPR enhanced the availability of macronutrients in the rhizosphere of infested soil. The antioxidant and defense enzymes in plant were significantly correlated with disease suppression. The PGPR can be used as a supplement with fungicides to combat adverse effect of disease.

The potential of three plants extracts, to protect potato plants against bacterial wilt caused by Ralstonia solanacearum was determined under greenhouse and field conditions. All soil drenching treatments of aqueous plant extracts of Hibsicus sabdariffa, Punica granatum and Eucalyptus globulus significantly reduced the disease severity compared with inoculated control. Although the applications of all three plant extracts resulted in similar reductions of disease severity in field up 63.23 to 68.39%, treatment of E. globulus leaf extract was found greater in restricting the symptom development than other the two plant extracts in the greenhouse. More than 94% reduction in the bacterial wilt symptom was observed in potato plants. All tested plant extracts were effective in inhibiting the growth of bacterial pathogen, not only in vitro, but also in stem of potato plants as compared with the inoculated control Potato plants treated with extract of H. sabdariffa reduced bacterial growth more effectively than treatment with P. granatum and E. globulus. Activity of defence-related enzymes, including peroxidase, polyphenoloxidase and phenylalanine ammonia lyase, were significantly increased in plants treated with the plant extracts compared to the control during the experimental period. In general, the higher enzymes activities were determined in both inoculated and non-inoculated treated potato plants after 8 days from plant extracts treatment. These results suggested that these plant extracts may be play an important role in controlling the potato bacterial wilt disease, through they have antimicrobial activity and induction of systemic resistance in potato plants.

Bacillus sp. BSp.3/aM, a beneficial rhizobacteria, was analyzed for the ability to improve plant health of chili by suppressing anthracnose disease. In the dual culture assay, the bacterium Bacillus sp. BSp.3/aM was found inhibitory to Colletotrichum capsica (6 mm). Further, upon seed priming, it reduced the seed-borne incidence of C. capsici (2%) and improved seedling vigor (1374 ± 7.15 vigor index) and germination (98 ± 0.57 %) of chili seedlings. Under greenhouse conditions, seed priming resulted in reducing the anthracnose disease incidence up to 20%. Induction of resistance against invading pathogen is through enhancing the activities of defense-related enzymes and higher accumulation of phenolic compounds in the host plant. The activity of phenylalanine ammonia-lyase (PAL; 95 units) was more at 48 hpi; peroxidase (POX; 6.49 units) at 24 hpi; polyphenol oxidase (PPO; 5.81 units) at 24 hpi and lipoxygenase (LOX; 9.9units) at 24 hpi. Maximum accumulation of the phenolics and chitinase accumulation was observed in BSp.3/aM + pathogen treated seedlings 120 hpi (94.7 μg/g tissue) and at 96 hpi (9.36 units), respectively. Thus, increased activities of defense-related enzymes (PAL, POX, PPO, LOX, and chitinase) correlated well with the decreased anthracnose incidence. Induced systemic resistance (ISR) mediated by PGPR was due to the upregulation of defense-related enzymes and by the accumulation of phenolic compounds.

Soybean (Glycine max L.) is a leading oil seed crop in the world. Owing to climate change, its production is challenged by many forms of biotic and abiotic stresses. Charcoal rot (Macrophomina phaseolina (Tassi) Goid) disease incidence is aggravated with the increase in soil and air temperatures. Charcoal rot disease in soybean is likely to gain its economic importance with the increase in global temperature. Apart from soybean, this pathogen has a wide host range including some economical crops like sorghum and maize. So far, complete resistance to this pathogen has not been identified in any of the crop species. Field screening techniques based on the colony-forming unit index (CFUI) and estimation of root stem severity (RSS) and glasshouse screening technique, such as cut-stem inoculation, are mainly employed in identifying charcoal rot resistance sources in soybean. High-throughput screening can be possible through cut-stem inoculation technique. There are reports indicating the correlation between field screening results and results obtained from this technique, and researchers have used this technique in understanding the genetic architecture of charcoal rot resistance and in identifying candidate genes and QTL governing charcoal rot resistance. Drought conditions are favourable for disease incidence and aggressiveness. Not all drought-tolerant genotypes are resistant to charcoal rot but some drought-tolerant genotypes are found to be moderately resistant to the disease. Significant yield losses are reported due to this disease even under irrigated conditions. Research is gaining momentum in developing high-throughput, reliable and repeatable glasshouse and in vitro screening techniques to identify stable sources of resistance and in understanding the genetic architecture of charcoal rot resistance. Breeding programs are under way for developing high-yielding, charcoal-rot-resistant and drought-tolerant cultivars.

Riboflavin caused induction of systemic resistance in chickpea against Fusarium wilt and charcoal rot diseases. The dose effect of 0.01 to 20 mM riboflavin showed that 1.0 mM concentration was sufficient for maximum induction of resistance; higher concentration did not increase the effect. At this concentration, riboflavin neither caused cell death of the host plant nor directly affected the pathogen`s growth. In time course observation, it was observed that riboflavin treated chickpea plants were inducing resistance 2 days after treatment and reached its maximum level from 5 to 7 days and then decreased. Riboflavin had no effect on salicylic acid (SA) levels in chickpea, however, riboflavin induced plants found accumulation of phenols and a greater activities of phenylalanine ammonia lyase (PAL) and pathogenesis related (PR) protein, peroxidase was observed in induced plant than the control. Riboflavin pre-treated plants challenged with the pathogens exhibited maximum activity of the peroxidases 4 days after treatment. Molecular weight of the purified peroxidase was 42 kDa. From these studies we demonstrated that riboflavin induced resistance is PRprotein mediated but is independent of salicylic acid.

Aim: Assessment of Cu-chitosan nanoparticles on controlling Bacterial disease by enhancing the activity of different antioxidant and defence enzymes in soybean.&#x0D; Study Design: Randomized block Method&#x0D; Place and Duration of Study: Department of Molecular biology and Biotechnology (MBBT), RCA, Maharana Pratap University of Agriculture and Technology, Udaipur Sub humid southern plain region of India between 2017-2019.&#x0D; Methodology: We synthesized, characterized and tested the efficacy of Copper-chitosan Nanoparticles against Bacterial Pustule disease in Soybean. Activity of defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)] were estimated after treating the plants with various concentrations of Cu-chitosan nanoparticles.&#x0D; Results: Three treatments of Cu-chitosan treatments (0.02, 0.06 and 0.10%) were found most efficient in disease control in both laboratory and net house condition) experiments. Cu-chitosan Nanoparticles application to soybean plants significantly enhanced the activity of defence enzymes (PAL and PPO) and antioxidant enzymes (SOD and POD) from 1.1 to 2.29 folds.&#x0D; Conclusion: Cu-chitosan NPs developed in this study were effective in controlling disease by enhancing the activity of different defence related enzymes [polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL)], and antioxidant [peroxidise (POD) and superoxide dismutase (SOD)]. This could be an important nonmaterial research in the field of agriculture due to better compatibility of biodegradable Cu-chitosan nanoparticles with biological control. In this study, the effect of Cu-chitosan nanoparticles to boost defence responses against Xanthomonas axonopodis pv. Glycine were evaluated.

Studies were conducted to develop the sustainable plant protection strategies against leaf blight of green gram incited by Macrophomina phaseolina by using plant extracts and fungicides under in vitro and in vivo conditions. In vitro studies indicated that plant leaf extracts of Adenocalymma alliaceum (77.20%) and Allium sp. (74.71%) demonstrated the highest inhibition of mycelia growth of M. phaseolina at 10% concentration. The results revealed the significant performance by foliar spray of carbendazim (0.1%), mancozeb (0.2%) and leaf extracts of A. alliaceum (10%), Allium sp. (10%) was more effective in reducing leaf blight disease in mungbean plants under greenhouse conditions. Further, induction of defense enzymes, such as peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), phenols and also chlorophyll content were studied. The enzyme accumulation was greater in treated plants as compared to untreated control. The present study revealed the induced systemic resistance (ISR) in enhancing the disease resistance in green gram plants against leaf blight disease by the application of plant extracts and fungicides. Key words: Macrophomina phaseolina, induced systemic resistance (ISR),Adenocalymma alliaceum, Allium sp.

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

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.

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.

The aim of the present study was to analyze induced expression of defense-related proteins in the soybean plants by rhizobacterial stain Carnobacterium sp. SJ-5 upon challenge inoculation with Fusarium oxysporum. Determination of the enzymatic activity of the different defense-related enzymes, phenylalanine ammonia lyase (PAL), lipoxygenase (LOX), peroxidase (POD) and polyphenol oxidase (PPO) was performed in the major parts of Glycine max L. Merrill using spectrophotometric method. Native-polyacrylamide gel electrophoresis analysis of the POD and PPO was employed followed by activity staining to find out the isoforms of respective enzymes. Activities of the PAL, LOX, POD and PPO were found to be highest in the bacterized root tissue of the soybean plants challenged with F. oxysporum. Isoform analysis revealed that PPO1, PPO4 and POD2 isoforms were expressed at higher levels in bacterized soybean root tissues challenge inoculated with the pathogen. Conclusively it was found that bacterial strain Carnobacterium sp. SJ-5 protect soybean plants from wilt disease caused by F. oxysporum by elicitation of the defense-related enzymes.

The findings of this study suggest that the selected five strains of Streptomyces spp. could be used for biological control of charcoal rot disease in sorghum. Two strains each of Streptomyces albus (CAI-17 and KAI-27) and Streptomyces griseus (KAI-26 and MMA-32) and one strain of Streptomyces cavourensis (SAI-13) previously reported to have plant growth-promotion activity in chickpea, rice and sorghum were evaluated for their antagonistic potential against Macrophomina phaseolina, which causes charcoal rot in sorghum. The antagonistic potential of these strains against M. phaseolina was assessed through dual culture assay, metabolite production assay, blotter paper assay in greenhouse and field disease screens. In both dual culture and metabolite production assays, the selected strains significantly inhibited the growth of M. phaseolina (63-74%). In the blotter paper assay, all the five strains of Streptomyces spp. inhibited the pathogen (80-90%). When these five strains were tested for their antagonistic potential under the greenhouse (two times) and field (two seasons) conditions by toothpick method of inoculation, significant differences were observed for charcoal rot severity. Principal component analysis capturing 91.3% phenotypic variations, revealed that the shoot samples treated with both Streptomyces and the pathogen exhibited significantly enhanced antioxidant parameters including superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase, phenylalanine ammonia-lyase, polyphenol oxidase, and total phenolic contents when compared to shoot samples treated with only M. phaseolina. Scanning electron microscope analysis revealed that the phloem and xylem tissues of the Streptomyces treated stem samples were intact compared to that of pathogen inoculated plants. This study indicated that the selected strains of Streptomyces spp. have the potential for biological control of charcoal rot disease in sorghum.