Evaluating the biochemical traits of novel Trichoderma-based biofilms for use as plant growth-promoting inoculants

Bacteria-mediated plant growth promotion is a well-established and complex phenomenon that is often achieved by the activities of more than one plant growth-promoting (PGP) trait, which may not always be present in a single organism. Biofilms developed using a combination of two organisms with useful plant growth-promoting rhizobacteria (PGPR) traits may provide a definite advantage. In this context, in vitro studies were conducted evaluating the PGP traits of novel biofilms developed using Trichoderma as matrix and agriculturally important bacteria (Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis) as partners. Such biofilms exhibited higher values for various biochemical attributes as compared to the individual organisms and dual cultures. Trichoderma–Bacillus and Trichoderma–Pseudomonas biofilms exhibited enhanced antifungal activity, ammonia, indole acetic acid (IAA) and siderophore production, as compared to the other treatments. Trichoderma–Azotobacter biofilm recorded the highest nitrogenase activity and 1-aminocyclopropane-1-carboxylic (ACC) deaminase activity. The synergism in terms of the PGP traits in the biofilms revealed their promise as superior PGP inoculants.     

Microbial Inoculants with Multifaceted Traits Suppress Rhizoctonia Populations and Promote Plant Growth in Cotton

The suppressive effects of microbial inoculants on cotton seedling mortality were assessed in Rhizoctonia solani‐infested soil. Per cent mortality ranged from 16 to 32 (60–120 days after sowing, DAS) and significant differences were recorded at 120 DAS, especially after drenching with compost tea of Azotobacter sp. and Anabaena torulosa—Trichoderma viride‐biofilmed formulations. The activity of hydrolytic enzymes was reduced in diseased root tissues due to a majority of the microbially inoculated treatments, compared with healthy root tissues. Per cent changes in the amounts of glomalin‐related soil proteins (GRSPs) were 2 to 85% greater than those of the uninoculated experimental controls. These microbial inoculants altered the rhizosphere bacterial communities as evident from the Denaturing gradient gel electrophoresis (DGGE) banding patterns and, also reduced the population of R. solani. While the copy numbers of the internal transcribed spacer (ITS) gene of R. solani in the uninoculated (infested soil) were approximately 1.47 × 10¹¹ per g soil, they were 1.34–1.42 × 10⁵ per g soil after the application of A. torulosa, Anabaena laxa and A. torulosa–Bacillus sp. Increases in yield (ranging from 3 to 23%) due to various microbial inoculants relative to uninoculated controls illustrated their promise as plant growth‐promoting and disease‐suppressing agents. This study illustrates the modulation of rhizosphere ecology through microbial inoculants as a mechanism of disease suppression and sustaining plant growth.     

Cyanobacterial diversity in the rhizosphere of rice and its ecological significance

This investigation was undertaken to characterize the abundance and genera-wise diversity of cyanobacteria in the rice rhizosphere and nitrogen-fixing ability of the isolated strains. The cyanobacterial strains belonging to the genera Nostoc and Anabaena comprised 80% of the rhizosphere isolates, which were also efficient in enhancing the germination and growth of wheat seeds and exhibited significantly high protein accumulation and IAA production. Distinct profiles for the cyanobacterial strains were obtained on amplification with extended Hip 1 primer — HipTG, indicative of the diversity among these strains. Our investigation helped in identifying promising cyanobacterial isolates from the rhizosphere of rice, which can be utilized in developing efficient plant growth promoting cyanobacterial inoculants.     

Soil and rhizosphere as habitats for Pseudomonas inoculants: new knowledge on distribution,activity and physiological state derived from micro-scale and single-cell studies

Pseudomonas spp. comprise an important group of bacteria used for biological control of microfungi in the plant rhizosphere. Successful performance of microbial inoculants requires both establishment, proliferation and activity under in situ conditions. To identify the factors controlling fate and performance of the inoculants, small-scale analyses are needed due to the heterogeneity characterizing the complex soil and rhizosphere environments. Direct staining techniques and advanced microscopy have provided the first detailed single-cell images of root colonization by these bacteria using fluorescent antibodies, fluorescent in situ hybridization and marker gene technology. These tracking methods have, in conjunction with activity assays, provided high-resolution data on the metabolic activity and growth of the inoculants. Finally, Pseudomonas reporter bacteria constructed to sense phosphorus, nitrogen, iron, and oxygen limitations have provided new insight into the significance of growth-limiting factors in the soil and along the root. The present work reviews the current knowledge on Pseudomonas inoculants in soil and rhizosphere based on these modern techniques. Finally, some perspectives for future studies are discussed.     

Effects of vanillin on the community structures and abundances of Fusarium and Trichoderma spp. in cucumber seedling rhizosphere

Soil microbial communities are crucial to the functioning of agricultural systems but little information is available on the effects of allelochemicals on soil microorganisms in vivo. Cucumber seedlings grown in soil were treated with different concentrations of vanillin (0.02–0.2?μmol/g soil), a phenolic compound with autotoxic activity. The community structures and abundances of Fusarium and Trichoderma spp. in cucumber rhizosphere were analyzed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis and quantitative PCR, respectively. Results showed that vanillin changed the community structures of Fusarium and Trichoderma spp. Vanillin decreased the number of bands of Fusarium spp., but increased the number of bands, Shannon–Wiener and evenness indices of Trichoderma spp. (p?Fusarium and Trichoderma spp. (p?Fusarium and Trichoderma spp., and that these two microbial groups showed different responses to vanillin.     

Characterization ofAzotobacter spp. and effect of Azotobacter and Azospirillum as inoculant on the yield and N-uptake of wheat crop

Out of ten isolates ofAzospirillum spp. isolated from the root and rhizosphere of wheat plant, seven belonged toA. brasilense and three belonged toA. lipoferum. All eight isolates ofAzotobacter spp. belonged toAzotobacter chroococcum. Two strains, one fromA. lipoferum and another fromA. chroococcum having high nitrogen fixing capacity with negative test for denitrification were used as inoculant to supplement the nitrogen need of wheat crop. Significant increases in the yield of wheat grain and uptake of nitrogen by the crop over the control were found in pot tests when the seeds were inoculated either withAzospirillum spp. orAzotobacter spp. or the combination of both the inoculants.     

<Emphasis Type="Italic">Nif</Emphasis>H-based studies on azotobacterial diversity in cotton soils of India

In order to promote the use of Azotobacter inoculants for cotton crop, a complete characterization of soil isolates of Azotobacter, isolated and screened on the basis of physiological properties, from four different cotton–wheat cropping regions of India was carried out, and their genetic diversity determined by RFLP (restriction fragment length polymorphism) analysis of the functional gene nifH. Genetic analysis of these isolates depicted a similarity coefficient of ≥80% among them, suggesting that though the isolates were obtained from different cotton soils of India, still they have large commonality in the nifH gene and constituted a homogeneous nifH population.     

Growth promotion ability of zoysiagrass rhizosphere fungi in consecutive plantings of wheat and soybean

Several isolates ofPhoma sp., certain nonsporulating fungi, as well asPenicillium andTrichoderma, all isolated from zoysiagrass rhizosphere, promoted growth of wheat and soybean under greenhouse conditions. However, the ability of these rhizosphere fungi to enhance plant growth varied with the crop tested. For example, most of the fungi effectively promoted the growth of wheat, whereas only a few fungi were effective on soybean. In consecutive plantings of wheat and soybean grown in soil previously infested with these zoysiagrass rhizosphere fungi, the growth promotion ability of the fungi was lowered. However, addition of fresh potting medium appeared to restore their growth-promotive effects. It appears that the activation of plant growth-promoting fungi in soil might depend on the availability of organic substrates to colonize, as evidenced by the promotion of plant growth.     

Soil Type Dependent Rhizosphere Competence and Biocontrol of Two Bacterial Inoculant Strains and Their Effects on the Rhizosphere Microbial Community of Field-Grown Lettuce

Rhizosphere competence of bacterial inoculants is assumed to be important for successful biocontrol. Knowledge of factors influencing rhizosphere competence under field conditions is largely lacking. The present study is aimed to unravel the effects of soil types on the rhizosphere competence and biocontrol activity of the two inoculant strains Pseudomonas jessenii RU47 and Serratia plymuthica 3Re4-18 in field-grown lettuce in soils inoculated with Rhizoctonia solani AG1-IB or not. Two independent experiments were carried out in 2011 on an experimental plot system with three soil types sharing the same cropping history and weather conditions for more than 10 years. Rifampicin resistant mutants of the inoculants were used to evaluate their colonization in the rhizosphere of lettuce. The rhizosphere bacterial community structure was analyzed by denaturing gradient gel electrophoresis of 16S rRNA gene fragments amplified from total community DNA to get insights into the effects of the inoculants and R. solani on the indigenous rhizosphere bacterial communities. Both inoculants showed a good colonization ability of the rhizosphere of lettuce with more than 10⁶ colony forming units per g root dry mass two weeks after planting. An effect of the soil type on rhizosphere competence was observed for 3Re4-18 but not for RU47. In both experiments a comparable rhizosphere competence was observed and in the presence of the inoculants disease symptoms were either significantly reduced, or at least a non-significant trend was shown. Disease severity was highest in diluvial sand followed by alluvial loam and loess loam suggesting that the soil types differed in their conduciveness for bottom rot disease. Compared to effect of the soil type of the rhizosphere bacterial communities, the effects of the pathogen and the inoculants were less pronounced. The soil types had a surprisingly low influence on rhizosphere competence and biocontrol activity while they significantly affected the bottom rot disease severity.     

Dominant fungi in the rhizosphere of established tea bushes and their interaction with the dominant bacteria under in situ conditions

Species of Penicillium and Trichoderma were found to dominate the rhizosphere of established tea bushes in a detailed study conducted from various tea growing locations in India. Penicillium erythromellis, P. janthinellum, P. raistrickii, Trichoderma pseudokoningii and T. koningii were found to be closely associated with tea roots. While seasonal fluctuation was observed in the case of Penicillium spp., the population of Trichoderma spp. showed less variation during the year. Both species were sensitive to low temperatures. In general, fungi associated with the tea rhizosphere were found to prefer a mesophillic temperature range (15 °C to 35 °C). The dominant species of Penicillium and Trichoderma also exhibited tolerance to lower temperatures, i.e., 5 to 10 °C on agar plates. Most fungi were able to grow in a wide range of pH (4 to 12). Lowering of soil pH in the rhizosphere of tea bushes was positively correlated with the age of the bush and may have affected the development of a specific microbial community in the rhizosphere.The populations of Penicillium and Trichoderma species were inversely correlated with the populations of two most dominant rhizosphere bacteria, Bacillus subtilis and B. mycoides. Both Bacillus species have been shown to have antagonistic activity against these two fungi under in vitro conditions. The present study demonstrates the existence of a similar antagonism under in situ conditions in the rhizosphere of established tea bushes.     

Effects of Acacia (Acacia auriculaeformis A. Cunn)‐associated fungi on mustard (Brassica juncea (L.) Coss. var. foliosa Bailey) growth in Cd‐ and Ni‐contaminated soils

Aims: To evaluate the effect of Acacia auriculaeformis‐associated fungi on the growth of mustard [Brassica juncea (L.) Coss. var. foliosa Bailey] in Cd‐ and Ni‐contaminated soils and design novel plant–fungi associations for bioremediation purpose. Methods and Results: Endophytic Trichoderma H8 and rhizosphere Aspergillus G16 were applied for rhizoremediation of Cd‐, Ni‐, and Cd–Ni combination‐contaminated soils through association with B. juncea (L.) Coss. var. foliosa. Compared with the noninoculated control plants, inoculation with Trichoderma H8 produced 109%, 41% and 167% more fresh weight (FW) plant yields in the Cd‐, Ni‐, and Cd–Ni‐contaminated soils, respectively (P < 0·05). Similarly, plants inoculated with Aspergillus G16 produced 109%, 47% and 44% more FW plant yields in these contaminated soils, respectively. Plants co‐inoculated with these two strains produced 118%, 100% and 178% more FW plant yields, respectively. The inoculations also increased the translocation factors and metal bioconcentration factors. Conclusions: The efficiency of phytoextraction for B. juncea (L.) Coss. var. foliosa was enhanced after inoculating with Acacia‐associated fungi. Significance and Impact of the study: The use of plant–fungi association may be a promising strategy to remediate metal‐contaminated soils.     

Optimization of conditions for in vitro development of <Emphasis Type="Italic">Trichoderma viride</Emphasis>-based biofilms as potential inoculants

Biofilms represent mixed communities present in a diverse range of environments; however, their utility as inoculants is less investigated. Our investigation was aimed towards in vitro development of biofilms using fungal mycelia (Trichoderma viride) as matrices and nitrogen-fixing and P-solubilizing bacteria as partners, as a prelude to their use as biofertilizers (biofilmed biofertilizers, BBs) and biocontrol agents for different crops. The most suitable media in terms of population counts, fresh mass and dry biomass for Trichoderma and Bacillus subtilis/Pseudomonas fluorescens was found to be Pikovskaya broth ± 1 % CaCO_3, while for Trichoderma and Azotobacter chroococcum, Jensen’s medium was most optimal. The respective media were then used for optimization of the inoculation rate of the partners in terms of sequence of addition of partners, fresh/dry mass of biofilms and population counts of partners for efficient film formation. Microscopic observations revealed significant differences in the progress of growth of biofilms and dual cultures. In the biofilms, the bacteria were observed growing intermingled within the fungal mycelia mat. Further, biofilm formation was compared under static and shaking conditions and the fresh mass of biofilms was higher in the former. Such biofilms are being further characterized under in vitro conditions, before using them as inoculants with crops.     

Studies on soil rhizosphere: speciation and availability of Cd

ABSTRACT

The rhizosphere soils of two durum wheat (Triticum turgidum var. durum L.) cultivars Kyle and Areola grown in two selected soils of southern Saskatchewan were collected both at 2-week and 7-week plant growth stages. The cadmium availability index (CAI), determined as M NH₄CI-extractable Cd, pH and the distribution of the particulate- bound Cd species of the soils were carried out and the data were discussed in comparison with those of the corresponding bulk soil. At the 2-week growth stage, the pH of the rhizosphere soil was less than that of the corresponding bulk soil and the CAI values were higher in the rhizosphere soil, indicating that more Cd was complexed with the low-molecular-weight organic acids (LMWOAs) at the soil-root interface and was extractable by M NH₄CI. Compared with the bulk soils, the CAI values were 2–9 times higher in the soil rhizosphere of the plots fertilized with Idaho monoammonium phosphate fertilizer at 2-week growth stage, which is attributed to the combined effects of the Cd introduced into the soil rhizosphere from the fertilizer (Cd content of the fertilizer was 144 mg kg^?1) and complexation reactions of phosphate and LMWOAs with soil Cd. At 7-week plant growth stage, such differences were not observed. The increased amounts of carbonate-bound and metal-organic complex-bound Cd species of the rhizosphere soils are due to the increased amounts of carbonate, a product of plant respiration, and the LMWOAs at the soil-root interface, respectively. Simple correlation analysis of the data showed that the CAI of the rhizosphere soils of the control plots correlated at least two orders of magnitude better with the metal-organic complex-bound Cd whereas the CAI of the rhizosphere soils treated with Idaho phosphate correlated better with carbonate-bound Cd species in comparison to other species.     

拮抗北细辛菌核病木霉菌的分离、鉴定及生防效果

[背景] 菌核病是北细辛根部主要病害之一,木霉菌作为目前应用最广泛的生物防治真菌,利用木霉菌防治北细辛菌核病是目前研究的热点。[目的] 通过稀释分离法对健康北细辛植株根际土壤进行菌株分离,以期筛选出有效拮抗北细辛菌核病的生防木霉菌。[方法] 以北细辛菌核病菌为靶标菌,采用平板对峙培养、挥发性与非挥发性物质抑菌的方法对分离得到的木霉菌进行筛选,采用生长速率法对筛选出的木霉菌的发酵液进行抑菌效果测定,并采用硫代巴比妥酸法测定筛选出的木霉对北细辛菌核病菌的丙二醛（Malondialdehyde,MDA）含量、紫外吸收法测定过氧化氢酶（Catalase,CAT）活性、氮蓝四唑法测定超氧化物歧化酶（Superoxide Dismutase,SOD）活性、愈创木酚法测定过氧化物酶（Peroxidase,POD）活性的影响。[结果] 从土壤中分离出木霉菌共14株,通过形态学和ITS-RPB2双基因联合构建系统发育树,鉴定其为哈茨木霉（Trichoderma harzianum）、钩状木霉（Trichoderma hamatum）、拟康氏木霉（Trichoderma koningiopsis）、深绿木霉（Trichoderma atroviride）、短密木霉（Trichoderma brevicompactum）和装絮木霉（Trichoderma tomentosum）。对峙培养试验表明,钩状木霉A26、拟康氏木霉B30、钩状木霉C6、哈茨木霉A17对北细辛菌核病菌抑制率均在90%以上,挥发性物质抑制测定结果显示钩状木霉C6抑制率最高,为53.73%±0.07%,木霉菌的非挥发性物质抑菌作用较强,哈茨木霉A17、钩状木霉A26、钩状木霉C6的非挥发性物质对细辛菌核病菌的抑制率均在75%以上,而拟康氏木霉B30抑制率可达100%。因此,筛选出的哈茨木霉A17、钩状木霉A26、拟康氏木霉B30、钩状木霉C6为拮抗效果较强的生防木霉菌,这4株木霉菌的发酵液对北细辛菌核病菌的抑制率分别为56.33%±0.12%、77.22%±0.06%、82.28%±0.03%、46.20%±0.04%。经这4株木霉菌的非挥发性物质处理7 d后,菌核病菌MDA含量显著增加,钩状木霉A26是对照组的7.7倍,最为显著;菌核病菌抗氧化酶活性均降低,与对照组相比,CAT、SOD、POD活性分别下降了19.67%-75.84%、4.71%-68.71%和3.57%-67.86%。[结论] 从北细辛健康植株根际土壤中分离的木霉菌株哈茨木霉A17、钩状木霉A26、拟康氏木霉B30、钩状木霉C6对北细辛菌核病菌均有较好的抑制效果,可用于北细辛菌核病的生物防治。     

Rhizosphere and root-infecting fungi and the design of ecological field experiments

As part of a wider study into the role of soil fungi in the ecology of the winter annual grass, Vulpia ciliata ssp. ambigua (Le Gall) Stace & Auquier, we applied the fungicides benomyl and prochloraz to three natural populations of the grass growing in East anglia, United Kingdom. The rhizosphere and rootinfecting fungi associated with the three populations were analysed each month between February and May 1992 when plants set seed. There were marked differences between the fungal floras associated with each of the three populations of V. ciliata, despite the fact that associated plant species and soil nutrient status were broadly similar between sites. This was attributed to wide differences in soil pH between the three populations. Prochloraz did not affect fungal abundance, but benomyl decreased the isolation frequencies of Fusarium oxysporum from roots and the frequencies of Penicillium and Trichoderma spp. isolated from rhizosphere soil, and increased the frequency of isolation of Mucor hiemalis from the rhizosphere of V. ciliata. There were also significant increases in the isolation frequencies of F. oxysporum from roots and M. hiemalis, Trichoderma spp. and Phoma fimeti from the rhizosphere of V. ciliata as plants matured. The significance of these results for the design of ecological field experiments are discussed in light of a previous study which has shown that asymptomatic root-infecting fungi can affect plant fecundity and hence abundance in natural populations of V. ciliata. We propose that differences in microbial communities between sites, controlled in part by soil chemistry, are a major factor determining plant performance under field conditions.     

Evaluating the promise of Trichoderma and Anabaena based biofilms as multifunctional agents in Macrophomina phaseolina-infected cotton crop

The influence of biofilmed formulations composed of Trichoderma viride and Anabaena torulosa as matrices was investigated in Macrophomina phaseolina (Tassi) Goid.-infected cotton crop, in terms of plant growth and biocontrol parameters. Trichoderma based biofilms were developed with Azotobacter chroococcum, Pseudomonas fluorescens and Bacillus subtilis, while A. torulosa biofilms were prepared using B. subtilis and T. viride as components. Scanning electron microscopy revealed significant colonisation of biofilms on the root surface, which could be correlated with lowest mortality of 5.67%, recorded using T. viride–B. subtilis biofilm. An increase of 4–7% in polyphenol oxidase enzyme activity was recorded in all biofilm-treated samples, particularly those in which B. subtilis was present. The highest value of 1360.22 µg microbial biomass carbon g^?1 soil was recorded in Anabaena–B. subtilis biofilm treatment. Significantly higher values of plant and soil nutrient parameters in treatments in which biofilms were used vis-à-vis individual cultures reveal their promise. Such novel biofilmed biofertilizers with multiple useful traits can be beneficial options for effective nutrient and pest management of cotton crop.     

Microbial changes in clover rhizosphere after foliar and soil application of cobalt

Application of cobalt(II) nitrate to the leaves of red clover (Trifolium pratense L.) resulted in a pronounced increase of dry weight and the number of root nodules. Counts of bacteria in the rhizosphere, content of ammonia and production of carbon dioxide m rhizosphere soil were also higher, whereas the content of nitrates decreased Differences m the counts of bacteria, aotmomycetes, Azotobaoter, anaerobic bacteria and cellulose decomposing bacteria m the rhizosphere of control and treated plants were not directly related to the way of application of cobalt. GeneraPenicillium, Fusarium andTrichoderma predominated among fungi. The relative occurrence of pemcillia was higher after the application of cobalt, the incidence of fusaria was lower. The effects of foliar and soil application of cobalt on rhizosphere microflora were not identical.     

Performance and persistence of phosphate solubilizingAzotobacter chroococcum in wheat rhizosphere

Survival and establishment of inoculant strains ofAzotobacter chroococcum NV 11 and its mutant NV 43 were assessed in sterilized soil and rhizosphere soil of wheat plants at 7 and 15 d interval, respectively, after sowing,i.e., up to 45 d under pot-house conditions. There was an apparent decrease in population of both strains in bulk soil but a steady increase was observed in root zones of the wheat plant. 10³ to 10⁴ introduced bacteria per g root were found sticking to roots. Further studies indicated that inoculant strains ofAzotobacter could survive, proliferate and establish well in root zones.     

溶磷细菌和丛枝菌根真菌接种对南方红豆杉生长及根际微生物和土壤酶活性的影响

以南方红豆杉实生苗为材料,采用盆栽实验探讨了高效溶磷细菌草木樨中华根瘤菌(Sinorhizobium meliloti)CHW10B与丛枝菌根真菌缩球囊霉(Glomus constrictum)单独和双接种条件下,其植株生长、根际土壤可培养微生物数量、土壤酶活和土壤微生物功能多样性变化,在微生态水平揭示接种对南方红豆杉生长的影响及其机制。结果表明:(1)各接种处理对南方红豆杉幼苗均有促生长作用,接种处理苗高、地径和生物量均较对照显著增加,并以双接种促进效果最好。(2)各接种处理提高了南方红豆杉根际土壤可培养细菌、真菌和放线菌含量,增加了土壤微生物碳源利用率,改变了土壤中物种的丰富度和均一度,增加了土壤中的生物多样性。(3)各接种处理促进了南方红豆杉根际重要土壤酶(酸性磷酸酶、脱氢酶、转化酶)活力的增加,且双接种的促进作用最为明显。可见,溶磷细菌(草木樨中华根瘤菌CHW10B)和丛枝菌根真菌(缩球囊霉)具有协同作用,两者同时接种可显著提高南方红豆杉根际土壤微生物数量及土壤酶活力,提高土壤微生物碳源利用率和土壤肥力,增加土壤中的生物多样性,从而达到间接促进宿主植物南方红豆杉生长的目的。