Colonization ability as an indicator of enhanced biocontrol capacity—An example using two Bacillus amyloliquefaciens strains and Botrytis cinerea infection of tomatoes

An understanding of biocontrol activities is important when developing microorganism‐based alternatives to conventional fungicides. From our bacterial collection, we selected two strains (BBC023 and BBC047) for their outstanding antagonistic capacity against fungal phytopathogens and growth‐promoting abilities towards Arabidopsis thaliana. According to physiological and molecular characterizations, both strains were classified as Bacillus amyloliquefaciens and were tested against Botrytis cinerea in vitro and in a tomato. Both strains secrete lipopeptide‐like compounds that contribute to their in vitro antagonism. SEM‐images showed altered B. cinerea mycelial structures that were consistent with previous reports of the direct action of lipopeptides against fungal hyphae. The strains were applied to the roots (R), leaves (foliar ‐ F) or root/leaves (R/F) on tomato plants. All treatments significantly reduced the severity of B. cinerea infection (measured as a control index). However, only root applications (R and R/F) led to growth promotion in the tomato plants. We detected the production of indole acetic acid (IAA) and 2,3‐butanediol as growth promotion traits in the two strains. For both strains, the R/F treatment showed the highest control index, suggesting a synergic effect of direct antagonism against B. cinerea and resistance induction in the plant. In addition, in vitro antagonism of BBC023 and BBC047 against B. cinerea was similar; whereas in the F application, strain BBC047 significantly improved plant resistance and maintained a higher population density over time on tomato leaves, compared to BBC023. BBC047 was also able to produce a complex and robust biofilm in Msgg medium compared with that of BBC023. We linked the reduced biocontrol of BBC023 on leaves with its limited ability to generate robust biofilms and colonize the phylloplane. At last, we highlight the potential of the native Bacillus strains as promising alternatives for the development of bioproducts for sustainable agriculture.     

In vitro antagonists of Rhizoctonia solani tested on lettuce: rhizosphere competence, biocontrol efficiency and rhizosphere microbial community response

The rhizosphere competence of 15 in vitro antagonists of Rhizoctonia solani was determined 4 weeks after sowing inoculated lettuce seeds into nonsterile soil. Based on the colonization ability determined by selective plating, eight strains were selected for growth chamber experiments to determine their efficacy in controlling bottom rot caused by R. solani on lettuce. Although in the first experiment all antagonists colonized the rhizosphere of lettuce with CFU counts above 2 × 10⁶ g⁻¹ of root fresh weight, only four isolates significantly reduced disease severity. In subsequent experiments involving these four antagonists, only Pseudomonas jessenii RU47 showed effective and consistent disease suppression. Plate counts and denaturing gradient gel electrophoresis (DGGE) fingerprints of Pseudomonas -specific gacA genes amplified from total community DNA confirmed that RU47 established as the dominant Pseudomonas population in the rhizosphere of inoculated lettuce plants. Furthermore, the DGGE fingerprint revealed that R. solani AG1-IB inoculation severely affected the bacterial and fungal community structure in the rhizosphere of lettuce and that these effects were much less pronounced in the presence of RU47. Although the exact mechanism of antagonistic activity and the ecology of RU47 remain to be further explored, our results suggest that RU47 is a promising agent to control bottom rot of lettuce.     

亚麻立枯病拮抗菌的筛选、生防效果及发酵条件优化

【目的】从健康亚麻植株的根际土壤中筛选对亚麻立枯病菌具有较强抑菌作用的拮抗菌,优化其产生抑菌活性物质的发酵条件,为其生防利用奠定基础。【方法】采用稀释平板涂布法和对峙培养法进行拮抗菌的筛选;根据菌株形态学特征、生理生化特性以及16S r RNA基因序列分析对其进行鉴定;利用温室抗病实验确定其生防效果;通过单因素实验和均匀设计实验优化其发酵条件。【结果】分离筛选到一株对亚麻立枯病菌具有显著拮抗作用的细菌HXP-5,且其对另外7种植物病菌真菌均有拮抗作用;鉴定菌株HXP-5为枯草芽孢杆菌;温室抗病实验结果表明其生防效果可达71.22%;其产生抑菌活性物质的最佳发酵条件为:葡萄糖为2.3%,胰蛋白胨+酵母粉(3:1)为0.25%,Na Cl为0.18%,发酵时间为72 h,发酵温度为27°C,转速为210 r/min,250 m L摇瓶装液100 m L,接种量为1.7%。【结论】经鉴定,对亚麻立枯病病菌具拮抗作用的菌株HXP-5为枯草芽孢杆菌,且对亚麻立枯病具有较强的防治效果,发酵条件进行优化后其对亚麻立枯病病原菌显示出更强的拮抗作用。     

Evaluation of two Streptomyces spp. and compost for growth promotion and biocontrol potential against Rhizoctonia solani on pepper

The individual and synergistic potentials of two Streptomyces strains and compost for growth promotion and biocontrol against dumping off caused by Rhizoctonia solani on pepper were evaluated. Results showed that the compost can greatly enhance pepper growth while the combination of the two strains and compost was most efficient for the disease suppression.     

Effect of UV‐C Radiation on Resistance of Romaine Lettuce (Lactuca sativa L.) Against Botrytis cinerea and Sclerotinia minor

The aim of this research was to examine the effect of UV‐C on resistance of lettuce to Botrytis cinerea and Sclerotinia minor. Analysis of the lesion surfaces showed that plants exposed to UV‐C were less susceptible to the two pathogens, especially on the fourth day after inoculation. Chlorophyll, carotenoid contents and malondialdehyde and hydrogen peroxide were assayed after 1 day and 4 days. Lettuces treated with UV‐C and inoculated showed an increase in chlorophyll and carotenoid content, especially 24 h after inoculation, and low values of the two indicators of oxidative stress as compared with lettuces which were inoculated but did not receive UV‐C treatment.     

Application of endophytic bacteria for the biocontrol of Rhizoctonia solani (Cantharellales: ceratobasidiaceae) damping-off disease in cotton seedlings

The antifungal potentialities of three endophytic bacterial strains, Stenotrophomonas maltophila H8 (Xanthomonadales: Xanthomonadaceae), Pseudomonas aeruginosa H40 (Pseudomonadales: Pseudomonadaceae) and Bacillus subtilis H18 (Bacillales: Bacillaceae) were evaluated against the phytopathogenic fungus Rhizoctonia solani in cotton seedlings under greenhouse conditions. The bacterial strains were applied as a soil drench or talc-based bioformulation in R. solani-infested soil and non-infested soil. Results indicated that the soil drench treatment was more efficient than talc-based bioformulation. A significant increase of seed emergence and seedling survival with a clear reduction of disease severity was achieved with the endophytic bacterial treatments. At the same time, the fresh weight, dry weight, shoot length and root length of the treated plants were markedly enhanced. Moreover, there was an apparent induction of the antioxidant enzymes (peroxidase, polyphenol oxidase and catalase) of the treated seedlings. Gas chromatography–mass spectrometry revealed the presence of various bioactive compounds in the bacterial supernatant. The antagonistic activity of the bacterial strains against R. solani was attributed to their capability to produce a broad spectrum of antifungal compounds in addition to bioactive molecules that can trigger the systemic resistance in the infected seedlings.     

Direct isolation of vacuoles from leaf tissue of lettuce (Lactuca sativa) retaining protoplasts within the leaves

A procedure is described in which vacuoles are isolated from leaf tissue of lettuce ( Lactuca sativa L.). After incubation in an enzyme solution, the vacuoles are directly extracted from the leaf tissue by osmotic shock using a phosphate buffer. In this method no protoplasts are released from the leaf tissue. This procedure avoids the problems of separating vacuoles from protoplasts with similar density. To evaluate the purity of the vacuoles, the activity of glucan synthetase 11 (EC 2.4.1.34), NAD(P) H-cytochrome c reductase (EC 1.6.99.3) and malate dehydrogenase (EC 1.1.1.37) was measured. To measure vanadate- and nitrate-sensitive ATPase activity (EC 3.6.1.8) vesicles were prepared from the vacuoles and ATP-dependent vesicle acidification was measured as acridine orange quenching. Nitrate inhibited the quenching, while addition of vanadate had no effect. It was concluded that the vacuoles were not contaminated with plasma membranes. To evaluate the viability of the vacuoles [¹⁴C]-malate uptake was measured. The vacuoles showed a constant rate of [¹⁴C]-malate uptake during 45 min. This rate was maximal at pH 6.8.     

Potential use and mode of action of the new strain Bacillus thuringiensis UM96 for the biological control of the grey mould phytopathogen Botrytis cinerea

The potential use of Bacillus thuringiensis UM96 as a biocontrol agent for the grey mould phytopathogen Botrytis cinerea was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, B. thuringiensis UM96 was characterised based on 16S rRNA and gyrA gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to B. cinerea, the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the chiB gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from B. thuringiensis UM96 showed a strong inhibitory effect of B. cinerea that was not observed with heat-treated supernatant. Interestingly, when the supernatant was supplemented with 100 µM allosamidin, a chitinase specific inhibitor, the antagonistic activity was suppressed significantly. A lack of chitinase activity was also observed in allosamidin-treated supernatants. Our pathogenic B. cinerea strain also exhibited susceptibility to pure Streptomyces griseus chitinase. Finally, the chitinolytic strain B. thuringiensis UM96 was able to protect Medicago truncatula plants in vitro from B. cinerea infection and significantly reduced the necrotic zones and root browning of the plants. Together, these results suggest a potential use of B. thuringiensis UM96 for the biological control of B. cinerea and a role for chitinases during the antagonism displayed.     

Development of an apparatus and protocol for the efficient isolation of bacteria and yeasts that attach to Botrytis cinerea germlings

An apparatus and protocol for the efficient and consistent isolation of bacteria and yeasts with the ability to attach to germlings of Botrytis cinerea is described. The study focused on minimising microbial contamination by bacteria or yeasts which do not attach to the pathogen B. cinerea but which interact with materials used in the equipment and would otherwise be isolated along with target microbes. After development, the assay reduced the contamination rate to 1–2 cells per 100 added and this was found to be a satisfactory level for the selection of microbial attachers to fungal hyphae. One or more phenotypes of microbes that adhered to B. cinerea germlings were found in 97% of the 70 samples collected from phylloplane washings and processed using this assay.     

Influence of bio-inoculants on the control of root rot and wilt disease of pyrethrum caused by Rhizoctonia solani

The effect of four bio-inoculants namely, Glomus aggregatum, Streptomyces sp., Bacillus subtilis and Trichoderma harzianum and a fungicide, Ridomil-mancozeb was evaluated on the biomass production and control of root rot and wilt disease under glasshouse conditions. Results showed that 21-day- prior inoculation with G. aggregatum was most effective where none of the treated plants produced disease symptoms and interestingly their growth was increased by 39.4%. The colonisation by G. aggregatum (>80%) also increased P concentration in shoot. While, similar treatment with Streptomyces sp., B. subtilis, T. harzianum and Ridomil-mancozeb individually failed to produce any significant effect over Rhizoctonia solani inoculated control, where all inoculated plant died, prematurely. The simultaneous and 3-day-post treatments of G. aggregatum were non-effective but simultaneous treatment with Streptomyces sp. produced 70% disease control, while B. subtilis and T. harzianum individually provided 50% control. Their effects were either better or at par when compared with the simultaneous treatment of Ridomil-mancozeb. In 3-day-post treatment, Streptomyces, B. subtilis and Ridomil-mancozeb individually provided 50% disease control, whereas T. harzianum was least effective as it could protect only 25% plants against infection. The results reveals that 15 days prior treatment of G. aggregatum can significantly controls the root rot and wilt disease of pyrethrum. Further, treatment of Streptomyces can also serve the next effective post infection control method.     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

Does the cutting of lucerne (Medicago sativa) encourage the movement of arthropod pests and predators into the adjacent crop?

Abstract  Lucerne ( Medicago sativa ) has been suggested as an ideal refuge habitat as part of an integrated pest management (IPM) program because it harbours high numbers of beneficial arthropods. Whether or not cutting of lucerne encourages the movement of these beneficials into adjacent target crops is unknown. Vacuum samples were used to determine the effects of cutting lucerne on arthropod abundance (pests and predators) within lucerne and adjacent soybean ( Glycine max ) crops. Vacuum-sample collections of arthropods were conducted before and after lucerne cutting on seven occasions in four fields over two seasons. In the lucerne, 10 m by 1 m strips parallel to the crop interface were sampled at 5, 10, 15, 20 and 30 m from the interface. In the soybean, 10 m of row were sampled at the same distances from the crop interface. The abundance of predators in lucerne was reduced immediately after cutting at all distances from the interface. Predator abundance in soybean did not show any change. The cutting of lucerne significantly reduced pest numbers within the lucerne but had little effect on pest abundance in the adjacent soybean. The temporal pattern in pest and predator abundance was very different for each field sampled. Generally, arthropods decreased in abundance after cutting and gradually increased as the lucerne grew back. In soybeans, arthropod numbers fluctuated regardless of the cutting of the lucerne. Cutting of lucerne alone does not guarantee movement of predators into the adjacent target crop. The presence of lucerne fields within a cropping area may have some impact on regional predator populations, and so still be useful for IPM programs, but this has yet to be tested critically.     

Identification of Botrytis cinerea genes up-regulated during infection and controlled by the Galpha subunit BCG1 using suppression subtractive hybridization (SSH)

The Galpha subunit BCG1 plays an important role during the infection of host plants by Botrytis cinerea. Delta bcg1 mutants are able to conidiate, penetrate host leaves, and produce small primary lesions. However, in contrast to the wild type, the mutants completely stop invasion of plant tissue at this stage; secondary lesions have never been observed. Suppression subtractive hybridization (SSH) was used to identify fungal genes whose expression on the host plant is specifically affected in bcg1 mutants. Among the 22 differentially expressed genes, we found those which were predicted to encode proteases, enzymes involved in secondary metabolism, and others encoding cell wall-degrading enzymes. All these genes are highly expressed during infection in the wild type but not in the mutant. However, the genes are expressed in both the wild type and the mutant under certain conditions in vitro. Most of the BCG1-controlled genes are still expressed in adenylate cyclase (bac) mutants in planta, suggesting that BCG1 is involved in at least one additional signaling cascade in addition to the cAMP-depending pathway. In a second SSH approach, 1,500 clones were screened for those that are specifically induced by the wild type during the infection of bean leaves. Of the 22 BCG1-controlled genes, 11 also were found in the in planta SSH library. Therefore, SSH technology can be successfully applied to identify target genes of signaling pathways and differentially expressed genes in planta.     

Induction of Reactive Oxygen Species and Phytoalexins in Onion (Allium cepa) Cell Culture by Biotic Elicitors Derived from the Fungus Botrytis cinerea

Metabolites of a phytopathogenic fungus Botrytis cinerea Pers. were analyzed for the presence of biotic elicitors. Three groups of elicitors competent in inducing defense responses inAllium cepa cells were identified and partly purified. The recognition of the elicitor signal in onion cells was shown to elevate the concentration of reactive oxygen species (ROS), namely, superoxide anion-radical (O₂^{\overset{-}.}) and hydrogen peroxide (₂₂). The intensity of ROS release depended on chemical identity of elicitor and its concentration. The most active ROS production in onion cells was induced by a protein fraction isolated from the medium for fungus culturing. The carbohydrate elicitors extracted from the fungus cytoplasm and cell walls of mycelia were much less effective. The dynamics of ROS generation comprised two stages. The first stage represented fast and low-amplitude changes that peaked in 15 min after the elicitor treatment. The second stage was more durable and extensive; it occurred in 1.5–6 h after the treatment.     

Resident bacteria, nitric oxide emission and particle size modulate the effect of Brassica napus seed meal on disease incited by Rhizoctonia solani and Pythium spp

Amendment of orchard soil with low-glucosinolate Brassica napus (rape) seed meal (RSM) suppresses infection of apple roots by Rhizoctonia solani but increases incidence of Pythium spp. infection. Following incorporation of Brassica sp. seed meals, soils were monitored for changes in populations of selected saprophytic and plant pathogenic microorganisms. When conducted in pasteurized soil, which possessed high numbers of Bacillus spp. and lower than detectable numbers of Streptomyces spp., RSM amendment did not provide control of R. solani. Populations of streptomycetes in RSM-amended soil increased to stable levels >20-fold higher than in non-amended soil. Disease suppressiveness was restored to pasteurized RSM-amended soil by adding any of several Streptomyces strains. Maximal rates of nitrification in orchard soil, determined by nitric oxide emission, were observed within two weeks following RSM amendment and inhibition of nitrification via application of nitrapyrin abolished the capacity of RSM to suppress R. solani infection of apple roots when seedlings were planted one day after soil amendment. Apple seedling mortality and Pythium spp. root infection were highest for seedlings planted immediately following incorporation of B. napus cv. Athena RSM, particularly when meal was added in a flake rather than powder form. Lower infection frequencies were observed for seedlings planted four weeks after RSM incorporation, even for soil in which densities of culturable Pythium spp. had not declined. Our results demonstrate that suppression of Rhizoctonia root rot in response to RSM amendment requires the activity of the resident soil microbiota and that initial disease control is associated with the generation of nitric oxide through the process of nitrification.     

Anastomosis Group Determination of Rhizoctonia solani Kühn (Telemorph: Thanatephorus cucumeris) Isolates from Tomatoes Grown in Aydin,Turkey and their Disease Reaction on Various Tomato Cultivars

In the Province of Aydin‐Turkey most frequently Fusarium spp. and secondly Rhizoctonia solani Kühn were isolated from the roots and crown of tomato plants. Based on affinities for hyphal fusion with test isolates, all R. solani isolates were identified as AG‐4. The tomato cultivars which were grown in soil infested with R. solani AG‐4 exhibited different reactions. From the point of symptom expression and the rate of seedling emergence Sunny 6066 F1 was found to be the most resistant cultivar, whereas Rio Grande, Rio Fuego, NDM 725, Interpeel and Konia were the most susceptible cultivars.     

碱性蛋白酶产生菌的筛选及发酵条件考查

从土壤中分离到的167株芽胞杆菌中选出一株高产、稳产碱性蛋白酶菌株A4－3（嗜碱性枯草芽胞杆菌）。该菌株最适产酶条件为（g／100ml）：蔗糖6．0;豆饼水解液10.0;Na2CO31．0;起始pH9．5。在该条件下,经37℃振荡培养48h,酶活力达2612u／ml。     

Seasonal growth of tree lupins (Lupinus arboreus) and the effect of defoliation on leaf production

The growth of tree lupins was investigated in two experiments. In the first, two ages of plant, 4-wk-old seedlings and 1-year-old plants, were transplanted into a ryegrass sward in an upland environment. Growth, in terms of leaf production, branching and stem elongation, was measured over two successive growing seasons. Plant dry matter and nutrient contents were determined at the beginning and end of each growing season. In the first summer, the rate of production of new leaves on the main stem of seedling plants averaged 1.8 leaves per wk and main stem length increased from 5 to 67 cm. On older plants, where floral apices had been initiated on main and primary stems, there was a 3–10 fold increase in secondary branch length. In the second season, there was no effect of plant age on rates of leaf appearance or stem extension; dry matter production was higher than in the first season. In the second experiment, the effect of removal of 0%, 50% or 100% of fully expanded leaves on the subsequent growth of 23-wk-old plants was investigated. During the 7-wk growth period, defoliation promoted the rate of production of mature leaves, and area and dry weight of new laminae were slightly higher in defoliated plants. Defoliation did not affect the concentrations of N, P or K in the new laminae, but P and K concentrations in petioles of defoliated plants were significantly higher than those in intact plants. The results from the experiments are discussed in relation to the potential use of tree lupins as nurse species and biomass crops in hill and upland environments of the UK.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.