Thermal sensitivity of Phytophthora cinnamomi and long-term effectiveness of soil solarisation to control avocado root rot

Root rot caused by the fungus Phytophthora cinnamomi is a major disease of avocados worldwide. Heat sensitivity of a collection of P. cinnamomi isolates was determined by exposing agar discs containing mycelium or mycelium plus chlamydospores at various temperatures for different periods. Long‐term effectiveness of soil solarisation to control Phytophthora root rot was evaluated in two field trials. In the first, soil disinfestation by solarisation was applied in 1990 to a naturally infested plot before planting avocado (Persea americana) and viñatigo (Persea indica) seedlings. In the second trial, established avocado trees were solarised for four consecutive summers (1996–1999). Results for heat sensitivity showed that fungal mycelium was inactivated after 1–2 h at 38°C. However, 1–2 h at 40°C was needed to kill all propagules when chlamydospores were present. Fungal growth inhibition after thermal treatments was related to levels of time and temperature, and detrimental effects occurred as consequence of sublethal thermal doses. Soil solarisation presented long‐term positive effects when applied as a preplanting treatment. Five years after solarisation, disease severity (0–5 scale where 0 = healthy and 5 = dead plant) of avocado and viñatigo planted in solarised soil was 2.03 and 0.71, respectively, compared with 4.65 and 4.84 in controls. Eleven years after solarisation, the percentage of dead plants in solarised soil was 73% for avocado and 43% for viñatigo but 100% in controls. In contrast, an insufficient level of control was observed in established orchards, probably because of the lower temperature reached during solarisation under the shade of tree canopy. In this situation, maximum temperatures at 5‐cm depth were 10–13.7°C lower than under solar‐heated mulch, only exceeding 40°C in 1997.     

Outbreak of Phytophthora cinnamomi causing severe decline of avocado trees in southern Turkey

Since the summer 2017, severe decline symptoms have been observed on 10- to 25-year-old avocado trees in almost all commercial orchards planted in the Mediterranean coastal region of Turkey. Young, newly planted trees in infected orchards were also affected by the disease. Affected trees showed wilting, leaf discoloration, defoliation and severe dieback. Some trees were completely desiccated. Although fine roots of symptomatic trees usually were decayed, reddish brown cankers also occurred on taproots and lateral roots of heavily infected trees. The pathogens were isolated from necrotic root and soil samples of symptomatic trees, using selective medium and soil baiting, and were identified based on morphological features and DNA sequences of the internal transcribed spacer (ITS) region. One isolate each of Phytophthora cryptogea and P. palmivora was identified, while all other isolates were P. cinnamomi. In addition, a subcortical fan-shaped mycelium, characteristic of Armillaria spp., was observed in the stem base of a symptomatic tree and identified as Armillaria gallica by DNA sequences of the internal transcribed spacer (ITS) and the translational elongation factor 1-α (EF 1-α) gene regions. Pathogenicity of Phytophthora isolates was tested by stem inoculation on one-year-old avocado seedlings. Two months after inoculation, canker lesions developed on stems of seedlings inoculated by any of the three Phytophthora spp. In contrast, collenchyma callus formed over the wound points on control plants over the same time period. This is the first report of P. cinnamomi, P. cryptogea, P. palmivora and A. gallica causing root rot of avocado trees in Turkey. In addition, P. cryptogea and A. gallica are reported for the first time associated with disease on this host. Due to the severe symptoms and widespread occurrence, P. cinnamomi should be considered a potential threat to avocado cultivation and natural ecosystems of this region of Turkey.     

Isolation, culture and regeneration of avocado (Persea americana Mill.) protoplasts

Protoplasts were isolated from embryogenic suspension cultures derived from avocado (Persea americana Mill.) zygotic embryos and nucellus in an enzyme digestion solution consisting of 1% cellulase Onozuka RS, 1% Macerase R10, 0.2% Pectolyase Y-23, 0.7 M mannitol. 24.5 mM CaCl₂, 0.92 mM NaH₂PO₄ and 6.25 2-[N-morpholino]ethanesulfonic acid (1.5 ml) mixed with 0.7 M MS^–8P (2.5 ml). MS^-8P medium consisted of Murashige and Skoog salts without NH₄NO₃, 1 mg l^–1 thiamine HCl, 100 mg l^–1 myo-inositol, 3.1 g l^–1 glutamine and 8P organic addenda. Medium osmolarity was adjusted with 0.15 M sucrose and 0–0.55 M mannitol. Protoplast yields of 3.5×10⁶ protoplasts g^–1 were obtained. Growth and development of the protoplasts were significantly affected by osmolarity, nitrogen source, plating density and culture medium dilution. Under optimum conditions, proembryos developed directly from embryogenic protoplasts and subsequently into somatic embryos. Optimum conditions for somatic embryo development included the culture of protoplasts at a density of 0.8–1.6×10⁵ ml^–1 in 0.4 M MS^–8P for 2–3 weeks, followed by subculture in 0.15 M MS^–8P at a diluted density of 20–40× for 1 month in darkness to obtain somatic embryos. Mature somatic embryos were recovered on semisolid medium; however, a low frequency of plantlet recovery (≤1%) from protoplast-derived somatic embryos was observed. Received: 9 February 1998 / Revision received: 4 May 1998 / Accepted: 15 May 1998     

Conidioma production of the white root rot fungus in axenic culture under near-ultraviolet light radiation

 Conidiomata of the white root rot fungus were produced in axenic culture under near-ultraviolet light radiation. Pieces of sterilized Japanese pear twigs were placed on 7-day-old oatmeal agar culture in plates. The plates were further incubated for 5 days and then illuminated by near-ultraviolet light. Synnemata developed on the twigs within 5 weeks in 19 of 20 isolates tested, and conidia were observed in 12 of the 19 isolates. The synnemata and conidia produced were morphologically identical to those of Dematophora necatrix. Received: October 29, 2001 / Accepted: March 11, 2002     

Environmental dynamics of Bacillus amyloliquefaciens CCMI 1051 antifungal activity under different nitrogen patterns

Aims: The aim of this study was to evaluate the influence of environmental conditions on the antifungal activity of the Bacillus sp. CCMI 1053 cultures. Methods and Results: The electrospray ionization mass spectra (ESI‐MS) analysis was used to detect the active peptides produced by Bacillus amyloliquefaciens CCMI 1051 cultures in a glucose‐containing medium to which four different nitrogen sources were added. The cultures produced different patterns of Bacillus sporulation and distinct antifungal activity of the cell‐free culture broths. Conclusions: The highest sporulation obtained corresponds to higher antifungal activity when it is formed after 3 days of microbial growth. The antifungal activity against Trichoderma harzianum CCMI 783 is more influenced by the concentration on the nitrogen source than the culture time of incubation. The association of nitrogen concentration and the time of incubation is particularly relevant in the expression of the antifungal activity. Significance and Impact of the Study: The present findings allow the reduction of the use of chemical pesticides and to limit some plant diseases. The association of the nitrogen source and the time of incubation is a novelty, which would improve the production of secondary metabolites. Both economical and environmental benefits arise from the study.     

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

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

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性

【目的】为得到枯草芽孢杆菌(Bacillus subtilis)G87的抗菌蛋白,明确其蛋白理化特性。【方法】采用硫酸铵沉淀和柱层析法进行分离纯化。【结果】获得单一抗菌活性蛋白(峰6-2-1),此抗菌蛋白分子量为50.8 kDa,等电点为5.90。经初步分析,抗菌蛋白不含脂,而含有少量(0.62%)糖;其蛋白部分具有脯氨酸或羟脯氨酸,但不含芳香族氨基酸。抗菌蛋白在高温(≥60℃)和较碱(pH8)环境下活性明显下降,但较抗紫外线、氯仿和胰蛋白酶、蛋白酶K、胃蛋白酶。【结论】枯草芽孢杆菌G87的抗菌蛋白为不含芳烃的糖蛋白,对高温和碱性条件敏感,而对蛋白酶类和紫外线等不敏感。     

Isolation and characterization of two hydrocarbon-degrading Bacillus subtilis strains from oil contaminated soil of Kuwait

Two strains of hydrocarbon-utilizing bacteria were isolated from soil samples of the Kuwait Burqan oil field at a temperature of 37 degrees C. The bacteria were motile endospore-forming rods with slight differences in their metabolic patterns and 16S rRNA sequence. Vegetative cells of the strains designated as AHI and AHII had an ultrastructure typical of gram-positive bacteria and showed gram-positive staining. The bacteria did not show pigmentation. Best growth was observed at 37 degrees C at neutral pH and NaCl concentrations in the range of 5-10 g per l. Both strains were obligatory aerobic and developed on synthetic media with either Diesel fuel, n-decan or naphthalene as the sole carbon and energy source. No specific growth factors were required. On the basis of their morphological, physiological and biochemical features, as well as their 16S rRNA analysis and electron microscope study, both strains were assigned to the species of Bacillus subtilis.     

Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria

Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species. Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate and characterize potent Bacillus strains with significant antagonistic activity against the major plant and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizosphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antagonistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates. Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA, phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture loss. The results of the present study could pave the way for developing potent drugs to combat microbial infection of plants and fish.     

Isolation and characterization of polymorphic simple sequence repeat loci in Armillaria ostoyae

A modified hybridization strategy was used to construct a microsatellite enriched library from DNA of Armillaria ostoyae, a serious root pathogen on pine. Sequence characterization of 19 random clones revealed 12 distinct loci harbouring a repetitive motif. Primer design from the flanking regions allowed for their development as polymerase chain reaction based markers. Polymorphic assessment at both the population and global levels revealed levels of variation useful for genetic studies. The level of cross‐species amplification observed with closely related Armillaria species was high, raising the possible exploitation of these primers across the genus.     

Isolation and characterization of ribosomes from Bacillus subtilis spores

Bishop, Helen L. (Syracuse University, Syracuse, N.Y.), and Roy H. Doi. Isolation and characterization of ribosomes from Bacillus subtilis spores. J. Bacteriol. 91:695-701. 1966.-The isolation of ribosomes from Bacillus subtilis spores was accomplished by freezing the spores in liquid nitrogen and grinding the spore pellet with an equal weight of levigated alumina. The ribosomes, which were adsorbed to the alumina, were freed by the addition of vegetative-cell ribosomes or bulk ribonucleic acid (RNA) to the crude alumina-ground extract. The spore ribosomes had sedimentation properties and RNA and protein compositions similar to those of vegetative-cell ribosomes. The difficulty encountered in obtaining spore ribosomes by ordinary extraction methods may be the result of nuclease and protease activities which were demonstrated in spore extracts.     

Degradation and solubilization of pectin by β-galactosidases purified from avocado mesocarp

Three β-galactosidase (EC 3. 2. 1. 23) isozymes were purified from the mesocarp of ripe fruit of Persea americana Mill. cv. Lula. and their effects on pectin derived from mature-green tomato fruit were investigated. The β-galactosidases had pl values of 5. 0,5. 1 and 5. 2. and molecular weights of 41. 49 and 54 KDa. respectively. There was a partial degradation of pectin resulting in the release of monomeric galactose upon treatment with avocado β-galactosidase. This degradation resulted in increased pectin solubility and decreased apparent average molecular size as determined by microfiltration and gel permeation by high-performance liquid chromatography. The increase in solubility was due. in part. to an apparent decrease in the ability of pectin molecules to aggregate together.     

The importance of avocado (Persea americana Mill.) fruits anthracnose and factors influencing the disease in Mana district,south-western Ethiopia

Anthracnose disease surveys were conducted in 25 farmers’ orchards, wholesaler and retailer shops in south-west Ethiopia. In addition, harvesting and postharvest practices, and storage conditions influencing disease development were studied with observation and questionnaire. The assessment results indicated significant variation among farmers’ orchards with the highest incidence (84.0 ± 16.7%) and severity index (26.0 ± 5.4%). Anthracnose damage of fruit was higher at retailers (76.7 ± 20.8%) than in the wholesalers shop (56.7 ± 32.5%). The total number of isolates identified was 249 and Colletotrichum gloeosporioides was the predominant pathogen proved by pathogenecity test. Among the major factors, harvesting avocado fruits with children (88%) and climbing on the tree (72%) resulted in fruit dropping that caused substantial injury and bruise. Generally, anthracnose caused by C. gloeosporioides of avocado fruit was prevalent in producer orchards that aggravated by traditional harvest and postharvest practices coupled to inadequate transportation and storage facilities at wholesaler and retailer shops with subsequent decay and loss of avocado fruits.     

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

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

Isolation and characterization of the lytic enzyme from Bacillus subtilis 797

The isolation procedure and some properties of the lytic enzyme produced by Bacillus subtilis 797 and capable of hydrolyzing the E. coli K-12 cells are described. Using hydrophobic chromatography on DNP-hexamethylene diamine Sepharose 4B and ion-exchange chromatography on DEAE-cellulose, a highly purified enzyme preparation with mol. weight of 28000, pI 8.2 has been obtained. The amino acid composition of the enzyme has been determined: Asp--37, Thr--17, Ser--34, Glu--15, Pro--14, Gly--17, Ala--36, Val--28, Met--4, Ile--11, Leu--17, Tyr--9, Phe--4, Lys--18, His--5, Arg--4. The enzyme is inhibited by a specific inhibitor of serine proteinases--benzylsulfonylfluoride, and is insensitive to EDTA and iodoacetic acid. The lytic enzyme has a proteolytic activity and splits the peptide substrate of bacterial serine proteinases--p-nitroanilide benzyloxycarbonyl-L-analyl-L-alanyl-L-leucine; the maxima for both activities lie within the pH range of 7.8-8.5. The lytic protease has the highest stability at pH 6-10. In some of its properties the enzyme is similar to serine proteinase from Bac. subtilis, i. e. subtilisins.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Isolation,characterisation of major secondary metabolites of the Himalayan Trichoderma koningii and their antifungal activity

Ethyl acetate extracts of two strains of Trichoderma koningii were evaluated for antifungal activity against soilborne pathogenic fungi, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum by poisoned food technique. Secondary metabolites, namely δ-decanolactone, 6-pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one were isolated from T. koningii (T-8) extract while palmitic acid, 6-pentyl-α-pyranone and stigmasterol were isolated from T. koningii (T-11) extract. Secondary metabolites were identified by ¹H NMR, ¹³C NMR and mass spectroscopic methods. These metabolites were evaluated for antifungal activity against the test pathogens 6-Pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one exhibited excellent antifungal activity against S. rolfsii. The antifungal activity though slightly less was comparable to that of hexaconazole, a commercial fungicide.     

β-oxidation enzymes and the carnitine-dependent oxidation of palmitate and palmitoyl CoA in mitochondria from avocado

Two sites for the β-oxidation of fatty acids in avocado (Persea americana L.) mesocarp exist. One site is the microbody, the other the mitochondrion. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose density gradient centrifugation, prevents rapid access of acyl CoA substrates to matrix β-oxidation sites. Thus, intact mitochondria showed little β-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of the acyl CoA substrates to matrix sites. Consequently, in ruptured mitochondria, high O₂-oxidation enzyme activities were measured. O₂ uptake studies further distinguished the two organellar sites of β-oxidation. During palmitoyl CoA oxidation, O₂ uptake was reduced by catalase and increased by KCN in the microbodies, whilst mitochondrial O₂ uptake was unaffected by catalase and reduced by KCN. This reflected the differing fates of FADH₂, produced during the first β-oxidation step, in the two organelles. In addition, only the mitochondrial β-oxidation of fatty acids was carnitine-dependent.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。