In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.     

<Emphasis Type="Italic">Halobacillus naozhouensis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from a sea anemone

A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071068^T, was isolated from a sea anemone (Anthopleura xanthogrammica) collected from the Naozhou Island on the Leizhou Bay in the South China Sea. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071068^T was able to grow with 1–20% (w/v) total salts (optimum, 6–9%), at pH values of 6.0–10.0 (optimum, pH 7.5) and a temperature range of 10–35°C (optimum, 25°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7 and the major cellular fatty acids were anteiso-C_15:0, anteiso-C_17:0 and iso-C_15:0. The genomic DNA G + C content was 42.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 071068^T belonged to the genus Halobacillus. The 16S rRNA gene sequence similarities between strain JSM 071068^T and the type strains of the recognized Halobacillus species ranged from 97.9% (with Halobacillus alkaliphilus) to 95.3% (with Halobacillus kuroshimensis). The levels of DNA–DNA relatedness between the new isolate and the type strains of H. alkaliphilus, Halobacillus campisalis, Halobacillus halophilus and Halobacillus seohaensis were 25.6, 22.1, 10.8 and 13.2%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 071068^T represents a new species of the genus Halobacillus, for which the name Halobacillus naozhouensis sp. nov. is proposed, with JSM 071068^T (=DSM 21183^T =KCTC 13234^T) as the type strain. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 071068^T is EU925615.     

新疆艾丁湖中度嗜盐苯酚降解菌多样性研究

高盐含酚废水属于极难处理的废水之一,筛选具有生物学降解能力的嗜盐菌有助于解决这一难题。从新疆艾丁湖盐湖中分离筛选能够降解苯酚的中度嗜盐菌,了解盐湖中度嗜盐苯酚降解菌的多样性组成和降解能力。研究结果表明,10%(质量分数)的盐浓度条件下,分离得到166株嗜盐菌,通过以苯酚为唯一碳源的培养基进行降解活性筛选后得到45株阳性菌,根据细菌16S rRNA基因序列系统进化分析,这45株菌分别归类到3个门,5个科,9个属。其中拟诺卡氏菌属(Nocardiopsis)是优势菌,占总量的68.8%,其余菌分布于Bacillus、Gracilibacillus、Pontibacillus、Halobacillus、Marinococcus和Halomonas属。在含100 mg/L苯酚的液体培养基,经过10 d培养后,这45株菌降解效率为1%~17%。本研究为工业应用提供了嗜盐微生物种质资源,极具进一步发掘和研究价值。     

Cloning and Characterization of the Genes for Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halobacillus dabanensis D-8T

A 11.2-kb fragment containing the ectABC genes of the biosynthetic pathway of ectoine from the Gram-positive, moderately halophilic bacterium Halobacillus dabanensis D-8^T was obtained by inverse polymerase chain reaction. Subsequently, the entire ectABC cluster was cloned and analyzed. It revealed that the intergenic regions of the ectABC genes from H. dabanensis D-8^T are more tightly spaced than those of Chromohalobacter salexigens, Halomonas elongata, Marinococcus halophilus, and Salibacillus pasteurii. The amino-acid sequence deduced from ectABC was highly homologous that from Virgibacillus pantethenticus (EctA 52%, EctB 60%, EctC 67%, respectively). The ectABC genes were cloned in the expression plasmid pMXB10 resulting in pMXB10ectABC. The ectoine was detected from cell extract in Escherishia coli ER2566 containing pMXB10ectABC using ¹³C nuclear magnetic resonance spectroscopy.     

Biomineralization of Carbonates by Marinococcus albus and Marinococcus halophilus Isolated from the Salar de Atacama (Chile)

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other Gram-positive bacteria and differs from that found in Gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10–100 μm, varying with salinity. They were of magnesium calcite (CO₃ Ca_1-x Mg_x) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates. Received: 3 November 1998 / Accepted: 9 March 1999     

Identification, phylogenetic analysis and characterization of obligate halophilic fungi isolated from a man-made solar saltern in Phetchaburi province, Thailand

A total of 17 species from 43 isolates were obtained through serial dilutions of soil samples isolated from one of the man-made solar salterns located in Ban Laem district of Phetchaburi province, Thailand. Soil analysis of the sample revealed high salinity and moisture content, slight alkalinity and low amounts of nitrogen, total organic carbon and organic matter in the habitat. Morphological analysis was performed on all isolates, and molecular identification and phylogenetic analysis were carried out only on the halophilic fungi isolated. Six halophilic fungi, belonging to four species, were identified among the isolates, including five strains of Aspergillus genus [Aspergillus flavus, A. gracilis, A. penicillioides (2 strains) and A. restrictus]. One species was found to be a yeast, namely, Sterigmatomyces halophilus, which was the most frequent isolate found among the halophilic fungi. All other isolates were halotolerant fungi. Characterization of the halophilic fungal isolates showed that they were best adapted to conditions of 10–15 % NaCl (w/v), slight alkalinity (pH 7.0–7.5) and a temperature range of 30–35 °C.     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

First report on the use of moderately halophilic bacteria against stem canker of greenhouse tomatoes caused byBotrytis cinerea

Experiments were conducted to study the effect of moderately halophilic bacteria isolated from different Tunisian Sebkhas (hypersaline soils), on stem canker caused byBotrytis cinerea on tomato plants grown under greenhouse conditions. Treatments performed with moderately halophilic isolates ofBacillus subtilis J9 andHalomonas sp. K2-5 significantly reduced stem lesion expansion byB. cinerea on tomato plants under greenhouse conditions. The use of such bacteria may constitute an important alternative to synthetic fungicides, which failed to suppress the development of the fungal pathogen.     

Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake,Iran

Screening of bacteria from different areas of Howz Soltan playa, a hypersaline lake in the central desert zone of Iran, led to the isolation of 231 moderately halophilic bacteria, which were able to grow optimally in media with 5–15% of salt, and 49 extremely halophilic microorganisms that required 20–25% of salt for optimal growth. These isolates produced a great variety of extracellular hydrolytic enzymes. A total of 195, 177, 100, 95, 92, 68, 65, 33, and 28 strains produced lipases, amylases, proteases, inulinases, xylanases, cellulases, pullulanases, DNases, and pectinases, respectively. In comparison with gram-negative bacteria, the gram-positive halophilic rods, showed more hydrolytic activities. Several combined activities were showed by some of these isolates. One strain presented 9 hydrolytic activities, 4 strains presented 8 hydrolytic activities, 10 strains presented 7 hydrolytic activities and 29 strains presented 6 hydrolytic activities. No halophilic isolate without hydrolytic activity has been found in this study. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the halophilic strains were identified as members of the genera: Salicola, Halovibrio, Halomonas, Oceanobacillus, Thalassobacillus, Halobacillus, Virgibacillus, Gracilibacillus, Salinicoccus, and Piscibacillus. Most lipase and DNase producers were members of the genera Gracilibacillus and Halomonas, respectively, whereas most of the isolates able to produce hydrolytic enzymes such as amylase, protease, cellulose (CMCase) and inulinase, belonged to gram-positive genera, like Gracilibacillus, Thalassobacillus, Virgibacillus, and Halobacillus.     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.     

Essential Oils as Biocides for the Control of Fungal Infections and Devastating Pest (Tuta absoluta) of Tomato (Lycopersicon esculentum Mill.)

Thymus capitatus and Tetraclinis articulata essential oils as well their major components (carvacrol and α‐pinene) were evaluated for their antifungal and insecticidal activities. Both oils showed good in vitro antifungal activity against Fusarium oxysporum, Aspergillus niger, Penicillium sp., Alternaria alternata, and Botrytis cinerea, the fungi causing tomato rot. In vivo results indicate the efficacies of both essential oils and carvacrol of reduce postharvest fungal pathogens, such as B. cinerea and Al. alternata that are responsible of black and gray rot of tomato fruit. Disease incidence of Al. alternata and B. cinerea decreased on average from 55% to 80% with essential oil of Th. capitatus and pure carcvacrol, while Te. articulata essential oil exhibited inhibition of fungal growth of 55% and 25% against Al. alternata and B. cinerea, respectively, with concentration of 0.4 μl/l air. The insecticidal activity of Th. capitatus and Te. articulata essential oils exhibited also a good insecticidal activity. At the concentration of 0.2 μl/ml air, the oils caused mortality over 80% for all larval stages of Tuta absoluta and 100% mortality for the first‐instar after 1.5 h only of exposure. α‐Pinene presented lower insecticidal and antifungal activities compared to essential oils of Th. capitatus, Te. articulata and pure carvacrol. Thus, these essential oils can be used as a potential source to develop control agents to manage some of the main pests and fungal diseases of tomato crops.     

Production of environmentally safe biocides from essential oils having antimicrobial activity

Different liquid formulations of anise, coriander and black cumin essential oils were used for preparing some biocides. The prepared formulations were tested for their antimicrobial activity against some post-harvest pathogenic microorganisms. The tested microorganisms were Fusarium oxysporum (Dray rot of potato), Alternaria alternata (Black rot of tomato), Penicillium italicum, P. digitatum, (Blue and green rot of orange, respectively), Botryitus cinerea (Gray rot of strawberry) and Erwinia carotovora (Soft rot of potato).The results revealed that the different formulations showed a complete inhibition effect on the growth of most of the tested microorganisms. Also, the antimicrobial activity of the formulated essential oils did not affect the formulation process compared with the original oil.     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Study of the efficacy of Aureobasidium strains belonging to three different species: A. pullulans,A. subglaciale and A. melanogenum against Botrytis cinerea of tomato

The difference in antagonistic activity against the causal agent of grey mould (Botrytis cinerea) of tomato between Aureobasidium strains belonging to three different species, namely A. pullulans, A. melanogenum and A. subglaciale, was evaluated by in vitro and in vivo assays. In the yeast–pathogen direct interaction experiment, all the strains significantly reduced B. cinerea growth, with A. melanogenum the least efficient species (17.8% of reduction) compared to A. pullulans and subglaciale (22 and 27.8%). The non-volatile metabolites produced by all three species reduced mycelial growth between 95 and 100%. These metabolites were characterised by FT-IR spectroscopy as polysaccharides, lytic enzymes, siderophores and antibiotics. The inhibitory effect of Aureobasidium strains on pathogenic enzymes such as xylanase, polygalacturonase and pectinase was measured showing A. pullulans strains as capable of strong inhibition of xylanase, an enzyme directly related to the virulence of necrotrophic pathogens such as B. cinerea. Our data demonstrate that the different species of Aureobasidium isolated from a range of non-conventional environments exerted variable efficacy against B. cinerea, with A. pullulans as the most active species followed by A. subglaciale and A. melanogenum as ineffective and not suitable for biocontrol applications.     

Phylogenetic analysis and screening of antimicrobial and cytotoxic activities of moderately halophilic bacteria isolated from the Weihai Solar Saltern (China)

A total of 45 moderately halophilic bacteria was isolated from sediment and saline water collected from the Weihai Solar Saltern (China). The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The halophilic strains were tested for their antimicrobial activity. Cytotoxicity assay was performed to determine which of the halophilic strains could inhibit proliferation of human hepatocellular carcinoma Bel 7402 cells. Our results showed that all of the isolated 45 strains displayed moderately halophilic characteristics. Phylogenetic analysis indicated that 17 of the isolated strains were related to the phylum Firmicutes and belonged to four genera, Bacillus, Halobacillus, Planococcus and Salinicoccus. The other strains identified as genus of Halomonas belonged to phylum γ-Proteobacteria. Most of the halophilic bacterial strains showed potent activities against Gram-positive bacteria, human pathogenic fungi and plant pathogenic fungi. In addition, the crude extracts from 14 halophilic bacterial strains showed cytotoxic activity against tumor cells Bel 7402, and five of them showed remarkable activities with IC₅₀ less than 40 μg ml⁻¹. Our results suggest that the moderately halophilic bacteria may be developed as promising sources for the discovery of novel bioactive substances.     

Evolution of bacterial flora from a subterranean saline well by graduated salinity changes in enrichment media

The bacterial flora of samples taken from a subterranean saline well was enriched (1) by periodic salinity increase and (2) by periodic salinity decrease, both at 25° and 35°C. During the enrichment process, bacterial flora, including halotolerant, marine, moderately and extremely halophilic bacteria, were enumerated. Results were similar at both temperatures. Marine bacteria and moderately halophilic microorganisms were the most favoured groups, predominating between 3 and 30% (w/v) salt content; extremely halophilic bacteria also appeared when salinity reached 30% (w/v) and halotolerant bacteria were poorly represented. The taxonomic distribution of 125 selected strains, chosen at random from counting media, was essentially similar to those from other hypersaline environments.     

Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.     

新疆地区盐湖的中度嗜盐菌16S rDNA全序列及DNA同源性分析

通过数值分类和16S rDNA PCR-RFLP分析,对分离自新疆地区的中度嗜盐革兰氏阴性菌进行研究,发现了一个新类群。在此基础上,进行了中心株AI－3的16S rDNA全序列分析,并与中度嗜盐菌已知种和相关种进行比较,得到系统发育树状图。在此树状图中,大多数参比菌株聚在一起,其16S rDNA全序列的同源性在96％以上,而AI－3与参比菌株的16S rDNA全序列相比,其相似性低于75％。但是,AI－3与Alcanivorax borkumensis^[1]的16S rDNA全序列的相似性为96％,与Halobacillus litoralis的16S rDNA全序列的相似性为99％,三者构成一个独立的发育分支。这说明在系统发育上,AI－3与参比菌株属于不同的分支,是一个新的类群。在新类群内,菌株之间的DNA同源性大于70％,而中心株AI－3与标准菌株伸长盐单胞菌（Halomonas elongata)的DNA同源性为44％,表明新分离的菌株可能构成一个新种群。