Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

Water Stress and Verticillium Wilt Severity on Eggplant (Solanummelongena L.)

Verticillium wilt is one of the most destructive diseases of eggplant (Solanum melongena L.). Some researchers have reported that wilt was encouraged by sufficient soil humidity, while others stated that it was encouraged by drought. This study investigated the water stress effect on the severity of Verticillium wilt on eggplant, as it is reflected on yield, agronomic traits and fruit quality. Thus, eggplant seedlings cv. ‘Tsakoniki’ were transplanted in three rows, each with 20 plants, during the summer of 1995 and 1996 in a plastic greenhouse, at the Agricultural Research Center of Macedonia and Thrace. Ten of the plants in each row were inoculated with the fungus Verticillium dahliae, while the other 10 were used as controls. Rows were irrigated every 2, 4 or 6 days. Soil humidity was calculated before every irrigation in each row. The disease severity was estimated by the disease index (DI) as the combination product of leaf symptom index (LSI) and vascular discoloration index (VDI). In addition, the plant height, early and total commercial yields, fruit numbers of early and total commercial yields, plant weight, the above-ground plant weight, root weight, pH, total soluble solids and fruit brilliance plus colour intensity were measured. The effect of Verticillium wilt on plants irrigated every 2, 4 or 6 days was estimated by the correlation coefficient (r) between LSI and DI and the aforementioned characteristics. Verticillium wilt had a significant but negative effect on all of the measured or calculated characteristics. This effect, however, was independent of the irrigation applied. On average, the early commercial yield was reduced by 40.8% and the final commercial yield by 39.4%. The only quality characteristic that was affected significantly by irrigation was the fruit brilliance and colour intensity (r =?0.640 to ? 0.727, P ≤ 0.01). Finally, the irrigation frequency (every 2, 4 or 6 days) had a significant but negative effect on all of the characteristics measured on the control plants. The only exception was fruit quality. In conclusion, the combined effect of irrigation and Verticillium wilt infection significantly reduced the early and total production of eggplant and spoiled the fruit quality.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Bacillus cereus CH2对茄子黄萎病的田间防治效果研究以及对根围微生态群落结构的影响

拟对Bacillus cereus CH2菌株在田间条件下,对茄子黄萎病的防治效果及其对茄子根围土壤微生物群落结构和功能的影响进行研究。田间试验显示蜡状芽孢杆菌Bacillus cereus CH2对茄子黄萎病有60.6%的防治效果。对菌剂和清水对照处理小区茄子根围土壤的Biolog分析显示,CH2的使用不会对根围土壤原有微生物群落结构和功能多样性产生显著影响,同时对六大类碳源利用能力的分析结果显示,CH2的使用能在植株生长前期提高根围土壤微生物群体对除酚类化合物的五大类碳源的利用能力。     

Optimal conditions for chitinase production by<Emphasis Type="Italic">Serratia marcescens</Emphasis>

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Molecular cloning and characterization of 58 kDa chitinase gene fromSerratia marcescens KCTC 2172

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from theSerratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids.Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide andN-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a 98% similarity to that ofS. marcescens QMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N′-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount ofN-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer (analogue of NAG₂), thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were 50°C and 5.0, respectively.     

Suppression of plant wilt diseases by nonpathogenic Fusarium oxysporum Fo47 combined with actinomycete strains

In order to build integrated strains with superior growth-promoting and disease-suppression effects, the biological control efficacy of Fo47 solid agents combined with actinomycetes strains toward Fusarium oxysporum and Verticillium dahliae were investigated in experiments on watermelon, cotton and eggplant. Five actinomycetes strains were prepared by solid fermentation. The count of viable solid agents, initially with propagules at 10⁷–10¹¹ CFU/g, slowly decreased after being stored one year at room temperature. After being inoculated into sterile soil for 50 days, the viable count of strain Fo47 remained at a stable level. The suppressive effects of Fo47 combined with strain QLP12 on Fusarium wilt on watermelon and cotton, and Verticillium wilt on eggplant, reaching 58.47%, 50.73% and 58.82%, respectively. This was significantly better than the single strain Fo47 alone, and growth of these treated plants and the colonisation rate of Fo47 were increased substantially as well. These results indicate that solid integrated agents with a high viability count and superior stability in soil could increase disease suppression and promote plant growth by synergy with different strains. The increased suppression obtained by Fo47 combined with actinomycete strains was not due to a simple addition of different mechanisms of biocontrol agents. By being intelligently integrated, these combinations increase disease suppression and provide the best biocontrol effect.     

Purification and properties of a chitinase from Penicillium sp. LYG 0704

The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be ¹AGSYRSVAYFVDWAI¹⁵. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).     

Purification and Characterization of Protease and Chitinase from Bacillus cereus TKU006 and Conversion of Marine Wastes by These Enzymes

A chitinase- and protease-producing bacterium was isolated and identified as Bacillus cereus TKU006. The better condition on our tests for protease and chitinase production was found when the culture was shaken at 25 degrees C for 2 days in 25 mL of medium containing 2% shrimp shell powder (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4).7H(2)O. The molecular masses of TKU006 protease and chitinase determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were approximately 39 and 35 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU006 protease and chitinase were 9, 50 degrees C, 3-11, 50 degrees C and 5, 40 degrees C, 3-11, 60 degrees C, respectively. TKU006 protease was inhibited completely by EDTA, indicating that the TKU006 protease was a metalloprotease. The TKU006 protease and chitinase retained 61%, 60%, 73%, and 100% and 60%, 60%, 71%, and 96% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 1 mM SDS, respectively. The antioxidant activity of TKU006 culture supernatant was determined through the scavenging ability on DPPH with 70% per milliliter. In conclusion, the novelties of the TKU006 protease and chitinase include its high stability to the surfactants and pH. Besides, with this method, we have shown that marine wastes can be utilized to generate a high-value-added product and have revealed its hidden potential in the production of functional foods.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Characterization and flocculating properties of a novel bioflocculant produced by <Emphasis Type="Italic">Bacillus circulans</Emphasis>

We studied a novel bioflocculant, PX, that is produced from Bacillus Bacillus circulans X3, and has excellent flocculating activity with regard to its characterization and flocculating properties. The bioflocculant was purified from supernatant by ethanol precipitation, dialysis and gel permeation chromatography (GPC). The major component of PX was an acid polysaccharide including uronic (19.8%), pyruvic (6.5%) and acetic acids (0.7%). It consisted of galactose, mannose, xylitol, rhamnose and galacturonic acid in an approximate molar ration of 5:4.1:3:2:1.2. The molecular weight of PX was about 4.85 × 10⁴ Da as determined by GPC. The infrared spectrum of the bioflocculant indicated the presence of carboxyl, hydroxyl, amino and methoxyl groups. Studies of the flocculating properties revealed that it was stable at 60–100°C and pH 4–10. Moreover, it could flocculate a kaolin suspension over a wide range of pH and temperature in the presence of CaCl₂.     

First report of <Emphasis Type="Italic">Verticillium tricorpus</Emphasis> isolated from potato tubers in Japan

In 1998, Verticillium sp. (CE98Vt1 and CE98Vt2) were isolated from discolored vascular structures of potato tubers sold at a market in Chiba Prefecture. These isolates were identified as Verticillium tricorpus on the basis of cultural and morphological characteristics and PCR diagnosis. This observed vascular discoloration of the potato tuber was demonstrated in three cultivars (Touya, Toyoshiro, and Waseshiro) among eight cultivars by inoculation to seedlings. External and internal symptoms of these isolates were not distinct in potato plants. The virulence of these isolates to potato was very low as compared with Verticillium dahliae. These two isolates were not pathogenic to Chinese cabbage, eggplant, green pepper, larkspur, parsley, snapdragon, soybean, tobacco, and tomato. This is the first report of V. tricorpus from potato in Japan.     

Highly selective biotransformation of ginsenoside Rb<Subscript>1</Subscript> to Rd by the phytopathogenic fungus<Emphasis Type="Italic"> Cladosporium fulvum</Emphasis> (<Emphasis Type="Italic">syn</Emphasis>.<Emphasis Type="Italic"> Fulvia fulva</Emphasis>)

Fourteen phytopathogenic fungi were tested for their ability to transform the major ginsenosides to the active minor ginsenoside Rd. The transformation products were identified by TLC and HPLC, and their structures were assigned by NMR analysis. Cladosporium fulvum, a tomato pathogen, was found to transform major ginsenoside Rb₁ to Rd as the sole product. The following optimum conditions for transforming Rd by C. fulvum were determined: the time of substrate addition, 24 h; substrate concentration, 0.25 mg ml⁻¹; temperature, 37°C; pH 5.0; and biotransformation period, 8 days. At these optimum conditions, the maximum yield was 86% (molar ratio). Further, a preparative scale transformation with C. fulvum was performed at a dose of 100 mg of Rb₁ by a yield of 80%. This fungus has potential to be applied on the preparation for Rd in pharmaceutical industry.     

Stereoselective epoxidation of <Emphasis Type="Italic">cis</Emphasis>-propenylphosphonic acid to fosfomycin by a newly isolated bacterium <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">simplex</Emphasis> strain S101

In industry, fosfomycin is mainly prepared via chemical epoxidation of cis-propenylphosphonic acid (cPPA). The conversion yield of fosfomycin is less than 50% in the whole process and a large quantity of waste is produced. Biotransformation by microorganisms is an alternative method of preparation. This kind of conversion is more delicate, environmentally friendly, and the conversion yield of fosfomycin would be higher. In this work, an aerobic bacterium capable of transforming cPPA to fosfomycin was isolated. The organism, designated as strain S101, was identified as Bacillus simplex by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. Fosfomycin was assayed by two means, bioassay and gas chromatography (GC). Glycerol was a good carbon source for growth and cPPA conversion of strain S101. When cPPA was used as the sole carbon source, neither growth nor conversion to fosfomycin occurred. The optimum cPPA concentration in the conversion medium was 2,000 μg ml⁻¹. After 6 days of incubation, the concentration of fosfomycin reached its maximum level (1,838.2 μg ml⁻¹), with a conversion ratio of 81.3%. Air was indispensable for the growth but not for the conversion to fosfomycin. Furthermore, vanadium ions were found to be essential for the conversion. High concentrations of cPPA had fewer inhibitory effects on the growth of strain S101.     

Production and partial characterization of chitinase from a halotolerant <Emphasis Type="Italic">Planococcus rifitoensis</Emphasis> strain M2-26

This paper is the first to investigate the production and partial characterization of the chitinase enzyme from a moderately halophilic bacterium Planococcus rifitoensis strain M2-26, earlier isolated from a shallow salt lake in Tunisia. The impact of salt, salinity concentration, pH, carbon and nitrogen sources on chitinase production and activity have been determined. This is the first report on a high salt-tolerant chitinase from P. rifitoensis, since it was active at high salinity (from 5 to 30% NaCl) as well as in the absence of salt. This enzyme showed optimal activity at 70°C and retained up to 82 and 66% of its original activity at 80 or 90°C, respectively. The activity of the enzyme was also shown over a wide pH range (from 5 to 11). For characterization of the enzyme activity, the chitinase secreted in the culture supernatant was partially purified. The preliminary study of the concentrated dialysed supernatant on native PAGE showed at least three chitinases produced by strain M2-26, with highest activity approximately at 65 kDa. Thus, the thermo-tolerant and high salt-tolerant chitinases produced by P. rifitoensis strain M2-26 could be useful for application in diverse areas such as biotechnology and agro-industry.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

Induction and purification of a thermophilic chitinase produced byAeromonas sp. DYU-too7 using glucosamine

In the presence of chitin,Aeromonas sp. DYU-Too7 can produce extra-cellular, chitin-degrading enzymes. Chitin analogues and other carbon sources can be used to cultivate this bacterial strain. The chitinases produced by the strain were higher in the GIcN (glucosamine) medium than those in other media. The maximal chitinase activity occurred in the medium containing 0.1% GIcN. Cultivation ofAeromonas sp. DYU-Too7 in the GIcN medium sped up the chitinase production; however the same result did not appear when it was cultivated in the (Chitin+GIcN) medium. This result may indicate that GIcN can be utilized byAeromonas sp. DYU-Too7 as a carbon source and an inducer to produce chitinases. A chitinase with a molecular mass of 36 kDa was further purified and characterized to have an optimal reacting pH of 5.0 and an optimal reacting temperature of 50°C. This chitinase showed high stability in the proximity of 30°C and also high stability in the proximity of pH 7.0. The hydrolysates of colloidal chitin, with the aid of the 36-kDa chitinase, were analyzed by an HPLC and found to be chitobiose.