Effect of environmental conditions and inoculum density on infection of guava fruits by Colletotrichum glososporioides

The influence of environmental factors (temperature and humidity), inoculum density on infection by Colletotrichum glososporioides and development of anthracnose lesions were determined on uninjured, sand-injured and punctured fruits. The optical temperature for severe infection was 30 °C, whereas the disease incidence was less at 20 and 35 °C. Inoculated guavas that received 1–60 h of continuous free moisture developed lesions, but the disease was minimal (0–7%) after 1–6 h free moisture. Infection rates of uninjured, sand-injured and punctured fruits receiving 60 h of free moisture were 34, 70 and 100%, respectively. Disease incidence increased as inoculum density increased from 101 to 106 conidia/ml. In field conditions, the development of anthracnose lesions was greater on punctured guavas than on uninjured or sand-injured ones, in both rainy and winter seasons. In general, the number of lesions was highest in sand-injured fruits, followed by punctured and uninjured fruits. In rainy season the number of lesions on injured and uninjured fruits was greater than similarly treated guavas in winter. This revised version was published online in June 2006 with corrections to the Cover Date.     

一株马兜铃内生真菌Colletotrichum sp.代谢产物中细胞毒活性成分的研究

从马兜铃内生真菌Colletotrichum sp.的大米发酵产物中分离得到6个化合物,经波谱数据分析分别鉴定为7-hydroxy-10-oxodehydrodihydrobotrydial(1)、格链孢酚(2)、5-甲氧基格链孢酚(3)、链格孢毒素I(4)、腾毒素(5)和二氢腾毒素(6)。以上化合物均为从该菌属中首次分离得到,其中化合物1～4对肺癌细胞和乳腺癌细胞有一定的细胞毒活性。     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Rapid Screening Method of Cassava Cultivars for Resistance to Colletotrichum gloeosporioides f.sp. manihotis

An in vitro method for assessing cassava anthracnose disease (CAD) resistance was developed as a preliminary screen to a CAD-resistant breeding programme. Potato dextrose agar (PDA) media was amended by extracts from the stem cortex of 10 cassava cultivars (30001; 30572, 30211, 88/02549, 88/00695, 88/01336, 91/00344, 91/00313, 91/00684 and 91/00475), and assayed for efficacy of inhibition of the growth of Colletotrichum gloeosporioides f. sp. manihotis isolates (05FCN, 10FCN, 12FCN, and 18FCN). Morphological and physiological data indicated that there was a significant difference (P ≤ 0.05), in mycelial growth, spore germination and sporulation among the four isolates on PDA amended with cassava stem extracts. Extracts from cassava cultivars 30211, 91/00684 and 91/00313 showed higher inhibition of germ tube development, mycelial growth and sporulation of the fungal isolates, whereas cultivars 88/02549 and 88/01336 showed the least inhibition. The 10 cultivars were further tested in both greenhouse and field conditions, under disease pressure for two planting seasons, to corroborate resistance to the fungus as observed in vitro . Greenhouse and field trials with the 10 cassava cultivars showed a significant difference ( P  ≤ 0.05) in CAD resistance. Cultivars 88/02549 and 88/01336 were highly CAD-susceptible, as shown in the in vitro assays and confirmed in the greenhouse and field tests. The other eight cultivars were either resistant (30211, 91/00684), or moderately resistant (30572, 88/00695, 91/00475, 91/00344, 30001 and 91/00313) to CAD. The study shows that an in vitro screening assay of cassava for resistance to CAD could serve as a convenient preliminary screening technique to discriminate CAD-resistant from CAD-susceptible cassava cultivars. The in vitro screening method considerably reduces time and labour in comparison with the current screening techniques of cassava, which involve field planting, inoculation and evaluation.     

Molecular Detection of Colletotrichum lindemuthianum by Duplex PCR

A duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5'-CTT TGT GAA CAT ACC TAA CC-3'; CY2: 5'-GGT TTT ACG GCA GGA GTG-3'), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare , not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species-specific primers of CD1/CD2 (CD1: 5'-ACC TGG ACA CAT AAG TCA AAG-3'; CD2: 5'-CAA CAA TGC CAG TAT CAG AG-3'). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR-based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.     

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.     

胶孢炭疽菌婆婆纳专化型侵染波斯婆婆纳的影响因子的研究

通过盆栽试验,研究了真菌自身生物学特性、杂草生长状况和环境因素等对胶孢炭疽菌婆婆纳专化型菌株QZ－97a侵染波斯婆婆纳的影响。结果表明,子叶期和衰老斯波斯婆婆纳感病性最强;培养7－14d后的孢子侵染力最强;病害发生的最佳温度范围是15－25℃;在保湿2－3d的条件下,露期中间的光照时间越短,病害发生越严重;保湿也可通过添加玉米粉和黄粉等介质来解决;达到理想致病效果的该菌株孢子悬浮液浓度必须在10^7个.ml^-1以上,由此可见,菌株QZ－97a在波斯婆婆纳发生时期的环境条件下可有效控制该杂草。     

PCR-based detection and characterization of the fungal pathogens Colletotrichum gloeosporioides and Colletotrichum capsici causing anthracnose in papaya (Carica papaya l.) in the Yucatan peninsula

Colletotrichum gloeosporioides is the common causal agent of anthracnose in papaya (Carica papaya L.) fruits, and infection by this fungal pathogen results in severe post-harvest losses. In the Yucatán peninsula (Mexico) a different Colletotrichum species was isolated from papaya fruits with atypical anthracnose lesions. The DNAs from a variety of Colletotrichum isolates producing typical and atypical lesions, respectively, were amplified by PCR with C.gloeosporioides-specific primers. All isolates from typical anthracnose lesions yielded a 450 bp PCR product, but DNAs from isolates with atypical lesions failed to produce an amplification product. For further characterization, the rDNA 5.8S-ITS region was amplified by PCR and processed for sequencing and RFLP analysis, respectively, to verify the identity of the papaya anthracnose pathogens. The results revealed unequivocally the existence of two Colletotrichum species causing anthracnose lesions on papaya fruits: C. gloeosporioides and C. capsici. PCR-RFLP using the restriction endonuclease MspI reliably reproduced restriction patterns specific for C. capsici or C. gloeosporioides. The generation of RFLP patterns by MspI (or AluI or RsaI) is a rapid, accurate, and unequivocal method for the detection and differentiation of these two Colletotrichum species.     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mm sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS–PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60°C and the optimum pH was 11.5–12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

珍珠番石榴果实发育及其矿质元素含量变化

本文探讨秋季开花的珍珠番石榴果实生长发育及其矿质元素含量的变化。结果表明,秋花春果的番石榴果实生长可分为三个阶段,即幼果较快生长期、生长停滞期和果实迅速膨大期。果实生长曲线呈双S型。随着果实生长发育,果实N、P、K、Ca、Mg相对含量降低,但其吸收总量逐渐增多,尤其在果实生长后期,对N、K养分需求剧增。     

Detection of latent infections caused by Colletotrichum sp. in olive fruit

Aims

To set up a practical method to detect latent infections of Colletotrichum sp., the causal agent of olive anthracnose, on olives before the onset of disease symptoms.

Methods and Results

Freezing, sodium hydroxide (NaOH), ethanol and ethylene treatments were evaluated to detect latent infections on inoculated and naturally infected olive fruit by Colletotrichum sp. as non‐hazardous alternatives to paraquat. Treatments were conducted using fruit of cultivars Arbequina and Hojiblanca. The disease incidence and T₅₀ were calculated. Dipping in NaOH 0·05% solution and the paraquat method were the most effective treatments on both inoculated and naturally infected fruit, although the value of T₅₀ was lower for the NaOH method than for the paraquat method in one of the experiments. Subsequently, the dipping time in NaOH 0·05% was evaluated. Longer dipping times in NaOH 0·05% were better than shorter ones in cultivar Arbequina, with 72 h being the most effective in cultivar Hojiblanca.

Conclusions

NaOH solution is a practical method to detect latent infections of Colletotrichum sp. on immature olive fruit.

Significance and Impact of the Study

This study is relevant because we set up a viable, non‐hazardous alternative to paraquat to detect latent infections of Colletotrichum sp. using NaOH. The use of NaOH is a simple and eco‐friendly tool that allows the determination of the level of latent infections by Colletotrichum in olives. Therefore, our method will be useful in decision‐making processes for disease management before the appearance of the first visible symptoms.     

Reaction of Some Coffea arabica Genotypes to Strains of Colletotrichum kahawae, the Cause of Coffee Berry Disease

Pathogenicity tests were performed on 11 genotypes of Coffea arabica using single‐isolate suspensions of Colletotrichum Kahawae obtained from 90 monoconidial isolates. The objective of this study was to estimate the proportion of pathogenic variation corresponding 10 differences in aggressiveness and virulence (races). A large part of the variation in the pathogen population was due to aggressiveness. The differential effects were too small to suggest conclusively that races exist. This paper discusses the possible causes for the observed small differential interaction and suggests breeding strategies that not only prevent possible adaptation of the pathogen to resistant varieties but also limit variation for resistance due to differences in aggressiveness of the pathogen.     

Toxic activity from liquid culture of Colletotrichum acutatum

Colletotrichum acutatum has become an increasingly important plant pathogen worldwide. With this background, a study was carried out to characterize the toxicity of liquid culture media from different isolates and to identify some properties of the toxic principles. Liquid culture media from all isolates were toxic to rubber leaves and induced the anthracnose symptoms. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Acetone soluble fraction of the culture filtrates retained the toxic activity and it was effective even at a concentration of 700 μg dry mycelium mass/ml. This revised version was published online in June 2006 with corrections to the Cover Date.     

Volatile organic compounds produced by Lysinibacillus sp. FJAT-4748 possess antifungal activity against Colletotrichum acutatum

FJAT-4748 is a bacterial strain isolated from forest soil samples taken from Dongba Valley, Lijiang, Kunming, Yunnan Province, PR China. This strain was identified as Lysinibacillus sp. based on a 16S rRNA gene sequence analysis. FJAT-4748 has been shown to possess antifungal activity against different fungi, including Colletotrichum acutatum, Aspergillus niger, Fusarium solani, Fusarium moniliforme and Fusarium oxysporum. The results of the present study indicate that this antifungal activity results from volatile organic compounds (VOCs) produced by this strain. The observed inhibition rates of VOCs from FJAT-4748 against these fungi were 100%, 100%, 37.20%, 18.94% and 7.64%, respectively. GC-MS analysis identified 24 VOCs from FJAT-4748, which included different categories of compounds, such as aldehydes, ketones, alcohols, aromatic hydrocarbons and alkanes. Of these 24 VOCs, the most abundant compound was 2-ethyl-1-hexanol, which constituted 36.24% of the total VOCs based on the relative peak area. In the in vitro C. acutatum mycelial growth assay, 2-ethyl-1-hexanol exhibited the strongest activity, with an inhibitory rate of 100% using 10?µL/plate of this VOC. The activity of benzaldehyde was lower. 2-decanone showed the weakest activity among the compounds tested. The inhibitory activity of an artificial mixture of three VOCs against the C. acutatum increased with the amount of artificial mixture used. The inhibition rate reached 100% using 30?µL/plate of this artificial mixture in the plate test. Taken together, these results show that the antifungal VOCs produced by Lysinibacillus sp. FJAT-4748 are potentially useful as agents for controlling anthracnose caused by Colletotrichum acutatum.     

A novel actin-related protein gene of Colletotrichum gloeosporioides f. sp. malvae shows altered expression corresponding with spore production

A novel actin-related protein (arp) was found in the plant pathogenic fungus, Colletotrichum gloeosporioides f. sp. malvae (Cgm), which causes anthracnose disease of round-leaved mallow (Malva pusilla). Sequence comparisons showed that this gene, arpA, belongs to the highly divergent 'other arps' category in the current arp classification system. ArpA is most similar to the arp11 gene of Mus musculus but has a unique structure with deletions at the C-terminus similar to that of the arp10 gene of Saccharomyces cerevisiae. A portion of another putative arp gene, arpB, was found immediately downstream of arpA. Expression of arpA was compared to the constitutively expressed Cgm actin gene, actA. In culture, the relative expression of arpA increased when growth conditions favored sporulation. During infection, arpA expression was greatest at the late necrotrophic phase, when sporulation occurred. Arps have been shown to be important in nuclear migration in fungal hyphae, and the expression pattern of arpA indicates that it may have a particular role during sporulation.     

Expression of chitin deacetylase from Colletotrichum lindemuthianum in Pichia pastoris: purification and characterization

The chitin deacetylase gene from Colletotrichum lindemuthianum UPS9 was isolated and cloned in Pichia pastoris as a tagged protein with six added terminal histidine residues. The expressed enzyme was recovered from the culture supernatant and further characterized. A single-step purification based on specific binding of the histidine residues was achieved. The purified enzyme has a molecular mass of 25 kDa and is not glycosylated as determined by mass spectrometry. The activity of the recombinant chitin deacetylase on chitinous substrates was investigated. With chitotetraose as substrate, the optimum temperature and pH for enzyme activity are 60 degrees C and 8.0, respectively. The specific activity of the pure protein is 72 U/mg. One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromol of acetate per minute under the assay conditions employed. The enzyme activity is enhanced in the presence of Co2+ ions. A possible use of the recombinant chitin deacetylase for large-scale biocatalytic conversion of chitin to chitosan is discussed.     

Molecular characterization and antimicrobial activity of endophytic fungi from coffee plants

Endophytic filamentous fungi from coffee plants (Coffea arabica and C. robusta) deposited in the Brazilian Collection of Environmental and Industrial Microorganisms (CBMAI) were characterized taxonomically by using molecular tools and investigated concerning their antimicrobial activity against different human pathogenic bacteria. Thirty-seven out of 39 CBMAI strains investigated were identified to at least at genus level by ITS and rDNA D1/D2 sequencing and phylogenetic analyses. Bioactivity screening of fungal extracts against Salmonella choleraesuis (CBMAI 484), Staphylococcus aureus (CBMAI 485), Pseudomonas aeruginosa (CBMAI 489) and against four different Escherichia coli serotypes showed that 17 fungi inhibited at least one of the bacteria studied. The endophytic fungi Trichoderma harzianum (CBMAI 43), Guignardia sp. (CBMAI 69) and Phomopsis sp. (CBMAI 164) inhibited from four to five bacterial species, while five fungi were active against all pathogenic bacteria tested and were identified as Aspergillus versicolor (CBMAI 46), Fusarium oxysporum (CBMAI 53), Glomerella sp. (CBMAI 63) and Cladosporium spp. (CBMAI 64 and CBMAI 66). The Minimal Inhibitory Concentration (MIC) for the fungus extracts varied from 0.025 to 1.0 mg ml⁻¹, demonstrating antimicrobial potential of some of these fungi.     

Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini

An improved procedure for the microbial hydroxylations of dehydroepiandrosterone (DHEA, 1) and 15 beta,16 beta-methylene-dehydroepiandrosterone (2) was studied using whole cells of Botryodiplodia malorum and Colletotrichum lini. C. lini catalyzed 7 alpha- and 15 alpha-hydroxylation of 1 and 7 alpha-hydroxylation of 2, while B. malorum gave 7 beta-hydroxylation of both the substrates. The stability of the enzymatic activity was higher in the presence of co-substrates (i.e., glucose or mannitol) allowing for repeated batches of the biotransformations. The yields of 7 alpha,15 alpha-dihydroxy-1 production were improved obtaining 5.8 gl(-1) (recovered product) from 7.0 gl(-1) of substrate. The structures of the hydroxylated products were assigned by a combination of two-dimensional NMR proton-proton and proton-carbon correlation techniques.     

内生真菌Colletotrichum sp. B501的寡糖提取物对黄花蒿发根中青蒿素生物合成的诱导(英文)

在黄花蒿 (ArtemisiaannuaL .)发根液体培养中 ,黄花蒿内生炭疽菌 (Colletotrichumsp .B5 0 1)细胞壁寡糖提取物可促进发根青蒿素的合成。经寡糖诱导子 (2 0mg/L)处理 4d后 ,发根青蒿素含量达 1.15mg/g ,比对照高出6 4 .2 9%。诱导作用与诱导子浓度、作用时间相关。诱导处理 1d后 ,X射线能谱分析表明黄花蒿发根细胞中Ca2 积累量显著增高 ,电镜观察发现液泡内出现高电子致密物 ,具活性氧清除作用的过氧化物酶表现出高活性 (6 .5unit·min-1·g-1FW)。诱导处理第三天 ,细胞核DNA呈梯度条带降解 ,部分细胞出现程序化死亡。内生菌细胞壁寡糖提取物引起的生理反应有利于细胞中青蒿素的生物合成。