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

Biocontrol and Plant Growth Promotion Characterization of Bacillus Species Isolated from Calendula officinalis Rhizosphere

The phenotypic and genotypic diversity of the plant growth promoting Bacillus genus have been widely investigated in the rhizosphere of various agricultural crops. However, to our knowledge this is the first report on the Bacillus species isolated from the rhizosphere of Calendula officinalis. 15 % of the isolated bacteria were screened for their important antifungal activity against Fusarium oxysporum, Botrytis cinerea, Aspergillus niger, Cladosporium cucumerinium and Alternaria alternata. The bacteria identification based on 16S r-RNA and gyrase-A genes analysis, revealed strains closely related to Bacillus amyloliquefaciens, B. velezensis, B. subtilis sub sp spizezenii and Paenibacillus polymyxa species. The electro-spray mass spectrometry coupled to liquid chromatography (ESI-LC MS) analysis showed that most of the Bacillus isolates produced the three lipopeptides families. However, the P. polymyxa (18SRTS) didn't produce any type of lipopeptides. All the tested Bacillus isolates produced cellulase but the protease activity was observed only in the B. amyloliquefaciens species (9SRTS). The Salkowsky colorimetric test showed that the screened bacteria synthesized 6-52 μg/ml of indole 3 acetic acid. These bacteria produced siderophores with more than 10 mm wide orange zones on chromazurol S. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. amyloliquefaciens (9SRTS) had no significant (P > 0.05) effect on the pre-germination of the chickpea seeds. However, it increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05).These results suggested that the Bacillus strains isolated in this work may be further used as bioinoculants to improve the production of C. officinalis and other crop systems. © 2013 Association of Microbiologists of India

In Vitro and In Vivo Characterization of Plant Growth Promoting Bacillus Strains Isolated from Extreme Environments of Eastern Algeria.

This report is to our knowledge the first to study plant growth promotion and biocontrol characteristics of Bacillus isolates from extreme environments of Eastern Algeria. Seven isolates of 14 (50 %) were screened for their ability to inhibit growth of some phytopathogenic fungi on PDA and some roots exudates. The bacteria identification based on 16S r-RNA and gyrase-A gene sequence analysis showed that 71 % of the screened isolates belonged to Bacillus amyloliquefaciens and the rest were closely related to B. atrophaeus and B. mojavensis. Most of them had high spore yields (22 x 108-27 x 108 spores/ml). They produced protease and cellulase cell wall-degrading enzymes while the chitinase activity was only observed in the B. atrophaeus (6SEL). A wide variety of lipopeptides homologous was detected by liquid chromatography-electrospray ionization-mass spectrometry analysis. Interestingly, some additional peaks with new masses were characterized, which may correspond to new fengycin classes. The isolates produced siderophores and indole-3- acetic acid phytohormone. The greenhouse experiment using a naturally infested soil with Sclerotonia sclerotiorum showed that the B. atrophaeus (6SEL) significantly increased the size of the chickpea plants and reduced the stem rot disease (P < 0.05). These results suggest that these isolates may be used further as bio-inoculants to improve crop systems