Dilip K. Arora

Fungal biotechnology in agricultural, food, and environmental applications

Agricultural biotechnology: a biotechnological approach to plant protection with fungi chemical identification of fungi - metabolite profiling and metabolomics isozyme analysis in fungal taxonomy, genetics and population biology molecular methods for identification of plant pathogenic fungi the application of molecular markers in the epidemiology of plant pathogenic fungi molecular biology in the control of mycotoxigenic fungi biotechnological potential of entomopathogenic fungi biotechnological potential of ergot alkaloids fungi as plant growth promoter and disease suppressor challenges and strategies for development of mycoherbicides biofungicides from microbial metabolites molecular biology of biocontrol trichoderma the biological control agent trichoderma - fundamentals to applications biotechnological strategies to control fungal diseases of vegetables control of postharvest fruits using microbes arbuscular mycorrhizal fungi in plant disease control commercialization of arbuscular mycorrhizal biofertilizer control of nematodes by fungi. Food and feeds: fungi in food technology - an overview role of fungi in fermented foods production of edible fungi mycoprotein and related high-value products from yeasts and fungi diversity and genetic variability of yeasts involved in wine fermentation yeast in the dairy industy flavours and aroma antifungal food additives molecular detection of fungi in foods and feeds the role of spoilage fungi in seed deterioration mycotoxin chapter genetics and biochemistry of toxin synthesis. Environmental biotechnology: cellulose degradation by fungi the importance of wood decay fungi in forest ecosystems the biodegradation of lignocellulose by white rot fungi biomineralization of heavy metals decolouration of industrial waste and degradation of dye water bioconversion of distillery waste by fungi degradation of hydrocarbons by yeasts and filamentous fungi biodegradation of azo dyes by fungi fungal degradation of explosives restoration of mycorrhizae in disturbed arid ecosystems.

Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil

The molecular diversity of aerobic endospore‐forming bacteria, typically Bacillus and its derived genera, has been investigated in various environments. However, there have been few investigations concerning Bacillus in acidic soils. In this study, the genotypic diversity and phylogenetic relationships among plant growth‐promoting (PGP) bacilli isolated from the rice rhizosphere growing in acidic soils of Kerala (pH varying from 6.3 to 6.8) were investigated. For assessing their biocontrol potential and PGP attributes, 115 isolates were randomly selected and 49 isolates that were positive for multiple traits were selected. Metabolic characterization of representative strains, using the Biolog GP2 (Gram Positive) MicroPlateTM, revealed a large versatility with respect to carbohydrate utilization. Amplified ribosomal DNA restriction analysis revealed 13 clusters at 65% similarity level, which consisted of 1–21 strains. 16S rDNA partial sequencing assigned all the isolates, except for one, to the Bacillus genus, with close relatedness to Bacillus humi, B. megaterium, B. drentensis, B. pocheonensis, B. aestuarii, B. arbutinivorans, B. niacini, and Brevibacterium casei. The Bacillus species with different metabolic capabilities, PGP abilities, and genetic diversity found in this study are likely to have ecological relevance. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Growth promotion of tomato plants by rhizobacteria and imposition of energy stress on Rhizoctonia solani

Rhizobacteria isolated from the rhizosphere of tomato plants were used to inoculate seeds and roots in greenhouse assays. Three bacteria, Azospirillum sp., Azotobacter chroococcum and Pseudomonas fluorescens, provided a significant (P = 0.05) increase in seedling emergence rate. A. chroococcum was found most effective in increasing the total dry weight, root and shoot length of tomato plants, followed by P. fluorescens and Azospirillum sp. Variations in incubation temperature and soil moisture significantly (P = 0.01) influenced the total plant growth. Temperatures >30°C were not favourable for bacterial activity or plant growth. Inoculated seedlings at −5 kPa (25°C) showed maximum plant growth, whereas least response was observed at −15 kPa. Inoculated seedlings significantly (P = 0.05) reduced the disease incidence and severity of Rhizoctonia damping-off of tomato seedlings. Colonization of Rhizoctonia solani sclerotia by rhizobacteria was negatively correlated with germination of sclerotia in patato dextrose broth (r = −0.84) and in Pfeffers salts solution (r = 0.85). Bacteria were able to release 14C-labelled endogenous compounds from labelled sclerotia. Sclerotia exuded 37% (percentage of total label) 14C-labelled carbonaceous compounds in soil inoculated with P. fluorescens, 29% with A. chroococcum and 19% with Azospirillum sp. Loss of endogenous C compounds from sclerotia was correlated (r = −0.97) with repression of germination.

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.

Pseudomonas aeruginosa inducing rice resistance against Rhizoctonia solani: production of salicylic acid and peroxidases.

Three isolates ofPseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice againstRhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) byP. aeruginosa bothin vitro andin vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King’s B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0–200 g/L showed no inhibition of mycelial growth ofR. solani in vitro, while at ≥300 g/L it inhibited it.P. aeruginosa-pretreated rice plants challenged inoculation withR. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungiR. solani, Pyricularia oryzae andHelminthosporium oryzae.

Genetic relatedness among and within different Rhizoctonia solani anastomosis groups as assessed by RAPD, ERIC and REP-PCR

The genetic relatedness among 41 isolates of Rhizoctonia solani belonging to 11 AGs was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by 3 RAPD primers (P14, R28 and RC09), ERIC (ERICIR-I/ERIC2) and REP (REP1R-I/REP2-I) gene sequences. Based on the banding patterns of PCR-amplified products, seven putative groups among the 41 isolates were recognized. RAPD-PCR generated multiple distinct products showing considerable variability among the isolates of different AG types. Isolates originated from the same geographical origin or host plants were not always genetically related. Amplification with ERIC and REP elements enabled detection of AGs/subgroup-conserved and isolate-polymorphic variants, respectively. Though pattern types generated by ERIC and REP in general, were distinctly variable among different subgroups, less variable fingerprint patterns were produced within the isolates of each subgroup.

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Abstract Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.

Antagonistic interactions between fungal pathogens and phylloplane fungi of guava

Dominant phylloplane fungi of guava (Psidium guajava L.) were screened for their antagonistic activities against the two pathogens,Colletotrichum gloeosporioides andPestalotia psidii, bothin vitro andin vivo. Culture filtrates ofAspergillus niger, Fusarium oxysporum andPenicillium citrinum caused more than 50% growth inhibition ofC. gloeosporioides. Filtrates ofCephalosporium roseo-griseum andF. oxysporum were most effective in reducing the growth ofP. psidii. Volatiles produced from the cultures ofA. niger, F. oxysporum, P. citrinum andP. oxalicum inhibited the growth ofC. gloeosporioides, whereas volatiles fromC. roseo-griseum, F. oxysporum andTrichoderma harzianum inhibited the growth ofP. psidii. The inhibitory effect of volatiles decreased with increase in incubation time. In general, the maximum effect of volatiles was noticed after 48 h incubation. Different grades of colony interactions in dual cultures were recognised between the two pathogens and the phylloplane fungi examined. Maximum inhibition ofC. gloeosporioides was caused byAureobasidium pullulans, Cladosporium cladosporioides, epicoccum purpurascens, F. oxysporum andMyrothecium roridum, whereasAspergillus terreus, C. roseo-griseum andP. oxalicum significantly reduced the growth ofP. psidii. Application of a spore suspension of each test fungus inhibited lesion development of guava leaves caused by the test pathogensin vitro. Inhibition was more pronounced when the spore concentration was increased.A. pullulans, C. cladosporioides, E. purpurascens, F. oxysporum, andT. harzianum were found to be strongly antagonistic toC. gloeosporioides. A. niger, A. terreus, C. roseo-grisem andT. harzianum were strongly antagonistic toP. psidii.

Cold‐active hydrolases producing bacteria from two different sub‐glacial Himalayan lakes

Microorganisms, native to the cold environments have successfully acclimatized their physiological, metabolic, and biological features, exhibiting uniqueness in their enzymes, proteins, and membrane structures. These cold‐active enzymes have immense biotechnological potential. The diversity of culturable bacteria in two different water lakes (the sub‐glacial freshwater and the brackish) of Himalayas was analyzed using SYBR green staining and cultural methods. A total of 140 bacteria were isolated and were grouped as psychrophiles, psychrotrophs, and psychrotolerant organisms, based on their optimal temperature for growth. The amplified ribosomal DNA restriction analysis using three restriction enzymes facilitated the grouping of these isolates into 96 genotypes at ≥85% polymorphism. Phylogenetic analysis using 16S rRNA gene sequences revealed that the bacterial strains from both lakes belonged to Firmicutes, Proteobacteria (α, β, and γ) or Actinobacteria. Screening of the germplasm for the activity of different cold‐active hydrolases such as protease, amylase, xylanase, and cellulase, revealed that about 16 isolates were positive, and exhibiting a wide range of stability at various temperature and pH. Our results suggest that the distinctly different ecosystems of sub‐glacial freshwater and brackish water lakes have diverse groups of bacteria, which can be an excellent source of extracellular hydrolases with a wide range of thermal stability.

Quantitative real-time PCR assay for rapid detection of plant and human pathogenic Macrophomina phaseolina from field and environmental samples

A real-time qPCR assay was developed to detect and quantify Macrophomina phaseolina abundance in rhizosphere soil and plant tissue. Both TaqMan and SYBR green techniques were targeted on ~ 1 kb sequence characterized amplified region (SCAR) of M. phaseolina and two sets of specific primers were designed for SYBR green (MpSyK) and TaqMan (MpTqK) assays. No cross-hybridization and no fluorescent signal exceeding the baseline threshold was observed in TaqMan and SYBR green assays, respectively. The minimum detection limit or sensitivity of TaqMan assay was 30 fg/μL of M. phaseolina DNA and limit of quantification of M. phaseolina viable population was estimated as 0.66 × 105 CFU/g soil−1 equivalent to 10 pg/μL of target DNA. This is the first report which demonstrated real-time qPCR assays with greater specificity and sensitivity to detect M. phaseolina population in soil and plant materials.