Segregation pattern of gene expression in cotton leaf curl virus-resistant transgenics

Non-pathogenic soil bacteria living in association with roots of higher plants enhance their adaptive potential and thus could be beneficial for their growth. Here, we present the current status of the use of Bacillus subtilis in biocontrol. Rhizobacteria are found in the rhizosphere. Plant growth promoting rhizobacteria (PGPR) strains, such as Bacillus and Pseudomonas, were isolated by using Nutreint dextrose Agar medium or Potato Dextrose Agar medium. The selection of PGPR strains was done by duel culture methods against the potato pathogens. The interaction of PGPR (Bacillus) with potato seeds or vegetative parts show promising antagonism by virtue of producing siderophore and antibiotics against black scurf and stem canker diseases of potato caused by Rhizoctonia solani, thereby resulting in increase of potato yield. The effectiveness of PGPR strain (Bacillus spp.) in improving the yield of potato in greenhouse conditions and in the field was observed.     

Comprehensive volatile organic compounds profiling of Bacillus species with biocontrol properties by head space solid phase microextraction with gas chromatography-mass spectrometry

The eight Bacillus strains, used as biocontrol agents with proven antagonistic effect against plant pathogens, produced antifungal volatile organic compounds (VOCs). Bioassay in sealed dishes revealed that the VOCs from each Bacillus strain significantly inhibited the mycelial growth (56–82%) of Fusarium solani. The effective antifungal VOCs were extracted using headspace solid phase microextraction and further identified by gas chromatography-mass spectrometry technique. The detected volatile compounds could be chemically grouped into ketones, alcohols, aldehydes, pyrazines, acids, esters, pyridines and benzene compounds. The ketones and alcohols were predominant in the VOCs from eight Bacillus strains whereas the ketones, including 3-methyl-2-pentanone, 2-heptanone, 2-octanone, 2-decanone, 5-methyl-2-hexanone, 2-nonanone, 2-dodecanone, 2-undecanone, 5-methyl-2-heptanoneand2-pentanone, were the most common and principal components in all strains. Present results showed that the eight Bacillus strains are rich resources of bioactive volatiles, which may play an important role in the inhibition on F. solani. Studies are under the way to determine effects of those compounds against plant pathogens and to find the possible action mechanisms.     

Screening tomato-associated bacteria for biological control of grey mold on tomato

Aiming at discovering effective biocontrol agents (BCAs) against grey mold on tomato caused by Botrytis cinerea Pers., we selected 819 bacterial isolates from the surface as well as the interior of the roots, stems, and leaves of tomato plants grown in B. cinerea-infested fields. In a dual-culture assay, 116 isolates (14.16%) showed antagonism against B. cinerea and fewer ones against five additional tomato-associated fungal pathogens – Pythium ultimum, Phytophthora capsici, Fusarium oxysporum f. sp. lycopersici, Sclerotinia sclerotiorum and Ralstonia solanacearum. Thirty-one isolates with antagonism to B. cinerea and at least one of the five additional pathogens were assessed for their efficacy in controlling grey mold on tomato in a greenhouse test. Thirteen of them attained the efficacy over 50% and were subjected to the second greenhouse test, in which 12 isolates consistently accomplished the biocontrol efficacy over 50%, with isolates ABc28 and ABc22 achieving the efficacy of 66.71% and 64.90%, respectively. Under greenhouse conditions, the above two as well as isolates ABc2, ABc11 and ABc17 increased tomato biomass by more than 20% in comparison with the control. The 12 antagonistic isolates accomplishing the biocontrol efficacy over 50% in both greenhouse tests were considered potential BCAs against grey mold, which were identified as Pseudomonas spp., Pantoea spp., Bacillus spp. and Chryseobacterium spp. Ten of them were found to produce at least one of the three hydrolytic enzymes (protease, cellulase and chitinase) and/or siderophore, which might be involved in their mechanisms of suppressing the disease. Based on the origin of these 12 strains, the leaf tissue, especially the leaf interior, of tomato plants grown in a B. cinerea-infested field appears to be a good source of potential BCAs against grey mold.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Antagonist effects of strains of Bacillus spp. against Rhizoctonia solani for their protection against several plant diseases: Alternatives to chemical pesticides

Rhizoctonia solan Kühn (teleomorph: Thanatephorus cucumeris (Frank) Donk (R. solani) is a soil-borne phytopathogenic species complex as well as a necrotic fungus that causes significant crop yield losses worldwide. Agronomic practices (crop rotation), resistant cultivars, and chemical pesticides are widely used to control R. solani. However, these practices are insufficient to control the pest. Moreover, the application of chemical pesticides is harmful to both the environment and human health. Therefore, the use of biocontrol agents (BCAs) and that of plant-growth promoting bacteria (PGPB) are considered to be potentially sustainable, cost-effective, efficient, and eco-friendly ways to control R. solani. Several microorganisms have been used as biocontrol agents (BCAs) to manage R. solani. Among these, biocontrol agents (BCAs) Bacillus spp. are used to promote plant growth. Furthermore, due to its broad range of antibiotic-producing abilities, Bacillus spp. is widely used against R. solani. In this review, current and previous studies about the ability of Bacillus spp. to control diseases caused by R. solani are reported. It also focuses on the plant-growth promotion attributes of Bacillus spp. in response to the deleterious effects of R. solani.     

Rhizotron studies on Zea mays L. to evaluate biocontrol activity of Bacillus subtilis

The effect of Bacillus as a biocontrol agent against some root-rot fungi was tested using maize (Zea mays L.) in rhizotrons placed in a growth chamber with relative humidity 60% with a 12 h photoperiod and day and night temperatures of 24 and 18°C respectively. Rhizoctonia solani Kühn caused pre-emergence damping-off in the untreated maize seeds showing weak and soft roots as observed through the perspex rhizotrons. Image analysis was used to quantify the effects of Bacillus treatment on seedlings infected with Pythium sp. Bacillus B77 and B81 were most effective in the control of the pathogen, R. solani which achieved a biocontrol activity of 24 and 35% respectively with regard to shoot dry biomass while B81 achieved 48% biocontrol with reference to root dry biomass. There was no effect on the root area. For root dry biomass, B81, B69, B11 and B77 showed higher biocontrol activity in comparison to the control. Pythium sp. caused pre- and post emergence damping- off in the untreated seeds. Root rot of the maize seedlings caused by Pythium sp. was slightly controlled by Bacillus B69 and B81 which achieved biocontrol activity of 18 and 11% respectively. For the biocontrol of Fusarium solani, Bacillus B77, B69, B81 achieved biocontrol activity of 50, 48 and 33% respectively with reference to root dry biomass.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Evaluation of isolates of Trichoderma,Pseudomonas and Bacillus species as treatment for the control of post-harvest fungal rot disease of yam (Dioscorea spp.)

The efficacy of four biological control agents (BCAs): Trichoderma asperellum strain NGT158, T. longibrachiatum strain NGT167, Bacillus subtilis and Pseudomonas fluorescens for the management of post-harvest tuber rot among four yam species, Dioscorea rotundata, D. cayenensis, D. alata, and D. dumetorum was evaluated. Rotted yam tubers were collected across three agroecological zones in Nigeria to isolate six infecting fungal pathogens: Aspergillus niger, Lasiodiplodia theobromae, Rhizoctonia solani, Penicillium oxalicum, Fusarium oxysporum and Sclerotium rolfsii. The BCAs were isolated by serial dilution and rot inhibition of treated tubers was evaluated using destructive sampling method in vivo after six months of storage. Bacillus subtilis was generally most effective, especially when applied 24 h before the inoculation of test pathogens across the four yam species, with percent inhibition that ranged between 47.8 and 81.2%. However, the four BCAs showed good potential in the control of the fungal pathogens causing post-harvest yam rot.     

Effect of Plant Age on Endophytic Bacterial Diversity of Balloon Flower (Platycodon grandiflorum) Root and Their Antimicrobial Activities

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of ‘low G + C gram-positive bacteria’ (LGCGPB) gradually increased 60.0–80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.     

Application of Soil‐borne Actinomycetes for Biological Control against Fusarium Wilt of Chickpea (Cicer arietinum) caused by Fusarium solani fsp pisi

Black root rot, caused by Fusarium solani f.sp. pisi, is a devastating soil‐borne disease in chickpea in Iran with no effective control measures. With the aim of finding applicable biocontrol agents to alleviate the malady, isolates of Actinomycetes isolated from soil and their antagonistic effect against F. solani f.sp. pisi were evaluated both in vitro and in vivo. More than 100 Actinomycetes isolates were screened for their antifungal activities against the pathogen. The most active isolates were evaluated in greenhouse for their biocontrol performance. Based on the results of dual cultures in screening evaluations, the size of inhibition zone of fungal growth, and the most effective antagonist isolates (S3, S12 and S40) were selected for further studies. Identity of active isolates was determined, in this regard, 16S rDNA of isolates were amplified using universal bacterial primers FD1 and RP2. The PCR products were purified and sequenced. Sequence analysis of 16S rDNA was then performed using NCBI BLAST method. Comparison of the near full length 16S rRNA sequence of isolates to GenBank sequences demonstrated that isolates S3 and S12 were most similar to Streptomyces antibioticus, while isolate S40 was most similar to Streptomyces peruviensis. Biocontrol studies of these isolates in control of the disease in greenhouse significantly decreased the disease severity. Actinomycetes isolate S12 demonstrated the greatest effect in reducing disease than the other two. Results of this research are at preliminary stage for developing biocontrol agents. These data can be utilized as a platform for future studies with the aim of commercializing these biocontrol products and hoping to step towards sustainable agriculture.     

Suppression of Phytophthora blight on pepper (Capsicum annuum L.) by bacilli isolated from brackish environment

Bacteria of the genus Bacillus are well known to possess antagonistic activity against numerous plant pathogens. In the present study, 11 strains of Bacillus spp. were isolated from a brackish environment and assayed for biocontrol activity under in vitro and in vivo conditions. Among the 11 isolates tested, nine isolates effectively inhibited the growth of various plant pathogens, namely Phytophthora capsici, Phytophthora citrophthora, Phytophthora citricola, Phytophthora sojae, Colletotrichum coccodes, Colletotrichum gloeosporioides, Colletotrichum acutatum, Rhizoctonia solani, Fusarium solani, Fusarium graminearum, Pyricularia spp., and Monilina spp. The effective isolates were further screened for suppression of Phytophthora blight of pepper plants under greenhouse conditions. The isolate SB10 exhibited the maximum (72.2%) ability to reduce the disease incidence and increased (32.2%) the vigour index of Capsicum annuum L. plants. Antifungal compounds produced by isolate SB10 were highly thermostable (100°C for 30 min). Matrix-Assisted Laser Desorption Ionization-Time of Flight mass spectrometry of the antifungal compounds revealed three lipopeptide complexes, namely the surfactins, the iturins, and the fengycins, which are well-known antifungal compounds produced by Bacillus spp.     

Medicago truncatula in Interaction with Fusarium and Rhizoctonia Phytopathogenic Fungi: Fungal Aggressiveness,Plant Response Biodiversity and Character Heritability Indices

Fusarium and Rhizoctonia genera are important pathogens of many field crops worldwide. They are constantly evolving and expanding their host range. Selecting resistant cultivars is an effective strategy to break their infection cycles. To this end, we screened a collection of Medicago truncatula accessions against Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani strains isolated from different plant species. Despite the small collection, a biodiversity in the disease response of M. truncatula accessions ranging from resistant phenotypes to highly susceptible ones was observed. A17 showed relative resistance to all fungal strains with the lowest disease incidence and ratings while TN1.11 was among the susceptible accessions. As an initiation of the characterization of resistance mechanisms, the antioxidant enzymes’ activities, at the early stages of infections, were compared between these contrasting accessions. Our results showed an increment of the antioxidant activities within A17 plants in leaves and roots. We also analyzed the responses of a population of recombinant inbred lines derived from the crossing of A17 and TN1.11 to the infection with the same fungal strains. The broad-sense heritability of measured traits ranged from 0.87 to 0.95, from 0.72 to 0.96, and from 0.14 to 0.85 under control, F. oxysporum, and R. solani conditions, respectively. This high estimated heritability underlines the importance of further molecular analysis of the observed resistance to identify selection markers that could be incorporated into a breeding program and thus improving soil-borne pathogens resistance in crops.     

A PCR-based method for the screening of bacterial strains with antifungal activity in suppressive soybean rhizosphere

Selection and evaluation of microbial strains for their antifungal activity in natural environments is time- and energy-consuming. We have adapted a PCR-based method to avoid these inconveniences. Soils that are naturally suppressive to plant disease were chosen as a source of antibiotic-producing bacteria. The screening was performed by means of PCR amplification using degenerate primers corresponding to peptide synthetase genes. Amplification fragments were obtained using template DNA from the rhizosphere of three different soybean fields. In order to assay their potential utility in pathogen control, several Bacillus strains were analysed for their in vitro antifungal activity by testing growth inhibition of Sclerotinia sclerotiorum. Four Bacillus sp. isolates gave a positive amplification signal, and three of them had an inhibitory effect on S. sclerotiorum growth, whereas two strains that failed to give an amplification signal did not inhibit fungal growth. These results show that PCR-based techniques could be useful to assess the presence of strains with potential use as biocontrol agents.     

Biocontrol of Rhizoctonia Damping-off of Tomato by Bacillus subtilis Combined with Burkholderia cepacia

Bacillus subtilis strain RB14‐C and Burkholderia cepacia strain BY were used in combination to control damping‐off of tomato plants caused by Rhizoctonia solani. Microcosm tests showed complete inhibition of R. solani growth on filter disks buried in soil added with the mixture of both bacteria. Single BY inhibited the fungus, but not completely, and RB14‐C had only slight inhibitory effect on pathogen growth. The efficacy of this combining treatment was checked in pot experiments, where bacteria were applied to the soil in several combinations: RB14‐C and BY together 4 days before seed planting, RB14‐C 4 days and BY 2 days before seed planting, RB14‐C 4 days and BY immediately before seeds. The effect of these treatments on population of R. solani in soil and infection of plants was compared with the activity of single application of each agent. All bacterial treatments significantly decreased damping‐off of tomato plants. The best control was obtained when BY was added 2 days after RB14‐C. In this treatment plant protection was significantly higher than that obtained in other combined applications and obtained by single strains, except BY added to the soil 4 days before seed planting. The lowest suppression indicated BY introduced to the soil before seed planting. RB14‐C only slightly decreased number of R. solani in the soil. In contrast, BY drastically reduced population of the pathogen. However, there was not a clear relation between decrease of pathogen density in soil and the rate of plant infection. The results show that combination of B. subtilis RB14‐C with B. cepacia BY can lead to greater damping‐off suppression than biocontrol exhibited by these strains used separately, but the effect of combining bacterial agents was clearly related to the order in which both agents were introduced.     

利用纤维二糖差向异构酶制备乳果糖的研究进展

乳果糖是由D-半乳糖和D-果糖两个基团通过β-1,4糖苷键连接而成的还原型二糖;乳果糖口服液具有治疗慢性便秘和肝性脑病的功效,在100多个国家作为常见非处方药(OTC)使用,需求量十分巨大;乳果糖还可以作为益生元改善人体肠道菌群关系。乳果糖的生产依赖化学法,其催化剂对人体有害,下游分离难度大。近年来,纤维二糖差向异构酶被发现能够高效催化乳糖制备乳果糖,该技术绿色环保、步骤简单,具有很强的产业化前景。本文结合自身研究经历对纤维二糖差向异构酶的研发情况进行总结,并综述了乳果糖酶法制备技术的现状。     

Use of Coniothyrium minitans and other microorganisms for reducing Sclerotinia sclerotiorum

Biological control agents (BCAs) Bacillus subtilis QST 713, Coniothyrium minitans CON/M/91-08, Streptomyces lydicus WYEC 108, and Trichoderma harzianum T-22 were evaluated for their efficacy in the reduction of survival of sclerotia and production of apothecia of Sclerotinia sclerotiorum under controlled environments. A growth chamber assay was conducted where 25 sclerotia were buried in pots containing potting soil, and BCAs were drenched into the soil at various concentrations, and five soybean seeds were planted in each pot. The presence and number of S. sclerotiorum apothecia were recorded daily. Sclerotinia sclerotiorum sclerotia were retrieved six weeks after seeding and viability was assessed on water agar plates. All BCAs were effective in reducing S. sclerotiorum inoculum at various efficacies. In general, efficacy was positively correlated with the rate of application. At the rate of application when the efficacy did not change significantly by increasing the rate, the BCAs had various reductions of apothecia and sclerotia. B. subtilis reduced apothecia and sclerotia by 91.2 and 29.6%, respectively; C. minitans reduced apothecia and sclerotia by 81.2 and 50%, respectively; Streptomyces lydicus reduced apothecia and sclerotia by 100 and 29.6%, respectively; Trichoderma harzianum reduced apothecia and sclerotia by 80.5 and 31.7%, respectively. In addition, the commercial strain of C. minitans CON/M/91-08, and a wild Michigan strain of C. minitans W09 were compared for their growth and sclerotial reduction. W09 had faster growth rate than the commercial strain, indicating potential diversities of biological control strains to be studied.     

Plant-growth-promoting traits and antifungal potential of the Bacillus amyloliquefaciens YL-25

Biological control of plant soil-borne diseases has been shown as an attractive and an environment friendly alternative to chemical fungicides. Different microbial strains have been reported effective in controlling plant pathogens. Among those, Bacillus strains have their own importance. Bacillus amyloliquefaciens strain YL-25, isolated from the rhizosphere of healthy banana plant, was evaluated as bio-organic fertiliser (BIO) for its ability to promote plant growth and suppress Fusarium wilt of banana in pot experiment. The results showed that the application of the BIO containing strain YL-25 significantly promoted the growth of banana plants and decreased the incidence of Fusarium wilt compared to the organic fertiliser and chemical fertiliser (CF). In order to explore the beneficial mechanisms of strain YL-25, experiments were conducted in vitro. The phytohormones including indole-3-acetic acid and gibberellin A3 and stable antifungal compounds three homologous of iturin A were identified in the culture broth of strain YL-25. The strain YL-25 also showed the ability to degrade extracellular phytate in plate experiment. Owing to its innate multiple functional traits and biocontrol activity, the strain YL-25 may be used as plant-growth-promoting rhizobacterium and biocontrol agent against Fusarium wilt of banana.     

Bacterial compounds,as biocontrol agent against early blight (Alternaria solani) and tobacco cut worm (Spodoptera litura Fab.) of tomato (Lycopersicon esculentum Mill.)

Abstract

Tomato, the important vegetable crop faces yield loses by foliar disease, Early Blight caused by Alternaria solani and infestation by tobacco cut worm, Spodoptera litura. Synthetic pesticides used for disease and pest control resulted in environmental pollution as well as development of resistance. By way of a need to develop a new biopesticide, bacteria were tested for their anti-fungal and insecticidal activity. Volatile compounds and peptides from the bacteria, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa were able to inhibit the hyphal growth and melanin production of A. solani. S. aureus showed the highest inhibitory effect as well as reduced the disease severity. B. subtilis exhibited the highest mortality rate of 87% and 83% against I and II instar of S. litura, respectively. The bacteria offered effective biocontrol at 35?°C. Bioactive substances from the bacteria can be used as potential biocontrol agents against the pest and disease of tomato.     

Molecular Characterization and Identification of Biocontrol Isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as “Trichoderma harzianum” Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

Isolation and characterization of a potential biocontrol agent Bacillus KM5 from rhizosphere soil of a rice plant

Twelve microbial isolates were isolated and purified from rhizosphere soil of a healthy rice plant. One of the isolate named as KM 5 showed antagonist activity in vitro against Sclerotium rolfsii Saccardo, Helminthosporium oryzae, Gibberella fujikuroi, Rhizoctonia solani Nees and Fusarium udum. KM 5 was characterized by microscopic, Gram stain and biochemical methods belonging to genus Bacillus. The genus Bacillus was further confirmed by isolation of genomic DNA, 16S rRNA amplification using Bacillus specific primers. Sequencing of the PCR amplified product using 775 base pairs and further NJ phylogenetic analysis confirmed that the microorganism is a new strain of bacterium named as Bacillus sp. KM 5. Partial sequence of 16S rRNA gene has been deposited to NCBI GenBank data base with accession no. EU266068 and deposited to MTCC with accession no. MTCC-5413. The cell free culture filtrate of Bacillus sp. KM 5 inhibited the growth of all test fungi. An antifungal protein produced by Bacillus sp. KM 5 was purified by ammonium sulphate. The protein was stable at 20, 40, 60 and 100°C and remained active after sterilization at 121°C for 15 min.