Secondary leaf fall of Hevea brasiliensis: factors affecting the production, germination and viability of spores of Colletotrichum gloeosporioides

The optimum temperature for growth and sporulation of Colletotrichum gloeosporioides from Hevea brasiliensis was between 26 and 32 ^oC, whereas spore germination exceeded 90% between 21.5 and 30.5 ^oC. Germination decreased in culture after 3 days, and on exposure of spores to sunlight or oven heat (46 ^oC) for 10 min. Spore viability and germination were sensitive to atmospheric humidity; at 99% r.h. germination was half that at 100% r.h. and was negligible below 97% r.h. Germination decreased by up to 30% after 3 h storage at 80% r.h. Continuous light favoured spore production in vitro, but spores produced in the dark had a higher percentage germination. No differences were detected between the numbers of spores germinating on leaves of different ages, although there were slightly more on susceptible cultivars and in the presence of extracts of uninfected susceptible leaves. Extracts from, infected leaves depressed spore germination, as did concentrations above 5 times 10⁵ spores/ml. The highest % germination was observed when naturally infected leaves were dry-stored for up to 20 days and then incubated for 2 days in a moist chamber.     

基于CRISPR-Cas9技术构建橡胶树胶孢炭疽菌的基因敲除系统

[背景]CRISPR-Cas9基因组编辑技术为病原真菌的基因敲除、敲入及定点编辑提供了新的思路。[目的]建立适用于橡胶树胶孢炭疽菌的CRISPR-Cas9基因敲除系统。[方法]通过大肠杆菌原核表达系统合成含有细胞核定位信号的Cas9蛋白;以URA5为靶标基因,预测该基因中Cas9的切割位点,并在体外转录合成相应的SgRNA;体外构建Cas9-SgRNA复合体,并将该复合体转入橡胶树胶孢炭疽菌原生质体;通过表型筛选及测序鉴定,筛选URA5的敲除突变体菌株。[结果]体外表达的Cas9蛋白与SgRNA能够形成复合体,并在体外对目标基因URA5的DNA序列进行切割;Cas9-SgRNA复合体能够成功转入橡胶树胶孢炭疽菌原生质体,并完成对URA5的敲除;敲除突变株表现出尿嘧啶缺陷表现型。[结论]建立了适用于橡胶树胶孢炭疽菌的基因敲除系统。     

Novel bacterial endophytes from Hevea brasiliensis as biocontrol agent against Phytophthora leaf fall disease

Bacterial endophytes offer control against many diseases of crop plants as potential biocontrol agents. Antagonistic bacterial endophytes acting against Phytophthora meadii have been screened from leaf, petiole and root tissues of Hevea brasiliensis. Six bacterial endophytes could exhibit more than 50 % inhibition of P. meadii, among which EIL-2, from disease-free zones showed a maximum of 62.5 % inhibition. The isolate EIL-2 was characterized as Alcaligenes sp. and the other isolates were identified as Pseudomonas aeruginosa. 16S rDNA sequence analysis showed that there existed genetic variation among the five isolates of P. aeruginosa from different tissues of the plant indicating the tissue type adaptation of the isolates. Dual culture technique with endophyte EIL-2 completely arrested the growth of P. meadii when inoculated prior to pathogen. The bioassay with EIL-2 in H. brasiliensis clones, RRII 105 showed 43 % reduction of lesion size on infected leaves whereas in RRIM 600 it was only 30 %.     

South American leaf blight of Hevea brasiliensis: spore dispersal of Microcyclus ulei

Trapping of ascospores and conidia of Microcyclus ulei among young trees of Hevea brasiliensis in Trinidad from May 1973 to May 1975 snowed that ascospores occurred throughout the year whilst conidia were present only during the wet season. Peak ascospore concentrations occurred in August and November during the wet season, the latter peak being more marked and the former coinciding with the period of maximum conidium liberation. In dry weather the number of ascospores increased during the night to a maximum at 06.00 h, and decreased to a low level during the day. On rainy days heavy ascospore discharge also occurred during the day. Ascospore concentration decreased significantly after dawn on sunny days whilst on overcast days the concentration remained high most of the day. Conidium production was highest around 10.00 h and decreased towards the evening to a low level during the night, reaching a minimum at 07.00 h.     

Identification and characterization of Neopestalotiopsis fungi associated with a novel leaf fall disease of rubber trees (Hevea brasiliensis) in Thailand

A novel leaf fall disease of rubber trees (Hevea brasiliensis) has been recently noted in Thailand. The fungal pathogens of this disease were identified based on both morphological and molecular characteristics as Neopestalotiopsis cubana and N. formicarum. Portions of internal transcribed spacer (ITS) region and the large subunit (LSU), translation elongation factor 1-α (TEF1-α) and β-tubulin (TUB) genes were PCR amplified with the primer pairs ITS1/ITS4, LR0R/LR5, EF1-728F/EF2 and T1/Bt2b, respectively. Sequencing of the PCR products and a phylogenetic tree based on the combined ITS, TEF1-α and TUB confirmed these pathogens as N. cubana and N. formicarum. Pathogenicity test results showed that the pathogens cause leaf spot and leaf fall similar to that observed in natural infections. This is the first report on the novel leaf fall disease of rubber trees in Thailand, with the results demonstrating that it is associated with N. cubana and N. formicarum.     

CgMFS1, a Major Facilitator Superfamily Transporter,Is Required for Sugar Transport,Oxidative Stress Resistance,and Pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis

Colletotrichum gloeosporioides is the main causal agent of anthracnose in various plant species. Determining the molecular mechanisms underlying the pathogenicity and fungicide resistance of C. gloeosporioides could help build new strategies for disease control. The major facilitator superfamily (MFS) has multiple roles in the transport of a diverse range of substrates. In the present study, an MFS protein CgMFS1 was characterized in C. gloeosporioides. This protein contains seven transmembrane domains, and its predicted 3D structure is highly similar to the reported hexose transporters. To investigate the biological functions of CgMFS1, the gene knock-out mutant ΔCgMFS1 was constructed. A colony growth assay showed that the mutant was remarkably decreased in vegetative growth in minimal medium supplemented with monosaccharides and oligosaccharides as the sole carbon sources, whereas it showed a similar growth rate and colony morphology as wild types when using soluble starch as the carbon source. A stress assay revealed that CgMFS1 is involved in oxidative stress but not in the fungicide resistance of C. gloeosporioides. Furthermore, its pathogenicity was significantly impaired in the mutant, although its appressorium formation was not affected. Our results demonstrate that CgMFS1 is required for sugar transport, resistance to oxidative stress, and the pathogenicity of Colletotrichum gloeosporioides from Hevea brasiliensis.     

Antifungal effects of dimethyl trisulfide against Colletotrichum gloeosporioides infection on mango

Colletotrichum gloeosporioides, one of the main agents of mango anthracnose, causes latent infections in unripe mango and can lead to huge losses during fruit storage and transport. Dimethyl trisulfide (DMTS) is an antifungal agent produced by several microorganisms or plants, but its effects on the infection process of C. gloeosporioides have not been well characterized. A histological investigation demonstrated that DMTS exhibits strong inhibitory effects on the infection process of C. gloeosporioides in planta by inhibiting the germination of conidia and formation of appressoria, damaging cytoplasm to cause cells to vacuolate and contributing to deformation of appressoria prior to penetration. This is the first study to demonstrate antifungal activity of DMTS against C. gloeosporioides on mango by suppression of the infection process, thus providing a novel postharvest biorational control for mango anthracnose.     

Ultrastructure and Differentiation of Protein-storing Cells in Secondary phloem of Hevea brasiliensis Stem

The ultrastructure and differentiation of the protein-storingcells in secondary phloem of terminal branchlets of Hevea brasiliensisMull. Arg. were studied using electron microscopy. The cellsare parenchyma tissue in the axial system and characterizedby the presence of a large amount of proteinaceous fibrils inthe central vacuole. The fibrils of the protein-storing cellsare straight, about 9 nm in diameter and arranged in a directionroughly parallel to the longitudinal axis of the stem. At theearly differentiation stage of the protein-storing cells, amass of proteinaceous fibrils appears in the cytoplasm, thenis separated from the peripheral cytoplasm by the endomembranesystem derived from endoplasmic reticulum, resulting in theformation of the central vacuole with the fibrils inside asthe vacuolar content The peripheral cytoplasm may continue toproduce proteinaceous fibrils with which the fibrils of thecentral vacuole is supplemented after the protein-storing cellsare formed. Hevea brasiliensis, storage protein, proteinaceous fibrils, vacuole, secondary phloem     

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.     

Evaluation of fungicides for control of South American leaf blight of Hevea brasiliensis

Of 43 fungicides tested in vitro, 19 showed strong, seven moderate and 17 weak inhibition of germination of conidia and ascospores of Microcyclus ulei. The formation of lesions on Hevea brasiliensis leaf discs was also suppressed by the first category of fungicides as well as by the five adjuvants tested. Ascospores were not released when perithecia were treated with urea, thiabendazole or alcoholic mercury chloride at 10.00, 0.10, 1.00 g/1 respectively; other fungicides had no such inhibitory effect. In field trials, thiophanate methyl (0.07% a.i.) and benomyl (0.025% a.i.) were most effective in controlling leaf infection, followed by chlorothalonil (0.15% a.i.) and mancozeb (0.32% a.i.). Benomyl suppressed conidial sporulation, whereas one application of thiophanate methyl (0.14% a.i.) to perithecia inhibited ascospore release; half of this concentration applied to conidial lesions or pycnidia caused the perithecia formed subsequently to abort. Thiophanate methyl thus shows promise for SALB control and elimination and benomyl may be valuable as a supplement in later rounds of spraying to control conidial sporulation. After 6 days of showery rain (2 mm for 17 min per day), water collected from sprayed leaves still gave complete inhibition of spore germination. However, inhibition was markedly reduced after 6 days of heavy rain (over 8 mm for 24 min per day).     

Agronomic and meteorological factors affecting the severity of leaf blotch caused by Mycosphaerella graminicola in commercial wheat crops in England

Factors affecting the severity of leaf blotch on the two upper leaves of wheat plants in crops at the milky ripe growth stage (GS 73–75) were investigated using survey data from 3513 randomly selected wheat crops sampled during 1985–1996. Year‐to‐year variation in disease severity was greater than spatial variability at county level, although both showed significant differences. The presence of disease above a 5% severity threshold was modelled using random effects logistic regression (Generalised Linear Mixed Model), which enabled risk variables measured at the field level to be combined with meteorological variables estimated at county level. The final model included terms for the fixed effects of disease resistance rating, date of sowing, high risk septoria periods in May and June, number of fungicide sprays and number of days with frost (≤‐2°C) in November. The percentage of crops above the threshold decreased with later sowing, increased number of November frost days and increased number of fungicide sprays. In contrast, high risk septoria periods (rain splash events) in May and June showed a positive correlation with the percentage of crops above the threshold. There were benefits from using resistant cultivars. The model showed that a range of risk variables were of broadly equivalent importance in determining the development of leaf blotch. These risk variables should be integrated in any scheme designed to support fungicide use decisions.     

Purification and partial characterization of phytotoxic glycopeptides secreted by Colletotrichum gloeosporioides

Colletotrichum gloeosporioides secretes a phytotoxic compound that causes anthracnose in Hevea brasiliensis: as a first step we attempted to follow mycelium growth, pH and toxin production by this fungus. The compound was isolated and partially purified by ultrafiltration, acetone precipitation and affinity and gel-permeation chromatography. Material purified was largely carbohydrate with a small protein fraction. Galactose, mannose and rhamnose were the neutral sugars encountered, and serine and threonine the major amino acids found. We propose a molecular mass for this substance of 50333 Da.     

蓝光诱导的胶孢炭疽菌(Colletotrichum gloeosporioides)类胡萝卜素积累

胶孢炭疽菌(Colletotrichumgloeosporioides)为一种丝状真菌,蓝光照射可诱导类胡萝卜素的积累。光镜下观察表明,蓝光可诱导胶孢炭疽菌菌丝积累色素颗粒,而黑暗和红光处理却无此现象。类胡萝卜素的积累受蓝光光照强度的影响。28℃且蓝光为6.5μmol.m-2.s-1时,类胡萝卜素积累量可随光照时间延长呈增长趋势,在第5天达到最高峰为71.8μg/g FW,随后含量下降。此外,胶孢炭疽菌在黑暗中预培养的时间也影响蓝光的诱导反应。     

Lipids of Hevea brasiliensis and Euphorbia coerulescens

The low MW lipids identified in the latices of Hevea brasiliensis and Euphorbia coerulescens were as follows: 2-methylcyclobutanone; 2-methyl-2-hydroxycyclobutanone; 2-methylcyclobutanol; euphol; euphorbol; and tirucallol.     

Effects of plant growth regulators and other compounds on flow of latex in Hevea brasiliensis

Eighty-nine compounds have been tested for their ability to promote the flow of latex from Hevea brasiliensis after tapping. The twenty-four substances which showed significant activity were: twelve chloro- and fluoro-phenoxy alkylcarboxylic acids substituted in the 4-, 2, 4- and 2, 4, 5- positions, two chlorinated benzoic acids, α- and β-naphthaleneacetic acid, 3-indolylacetic acid, 2-methoxy-4-diethylamino-6-isopropylamino-1, 3, 5- triazine (methoxy-ipazine), N,N-diallyl-α-chloroacetamide (CDAA), 1,1′-dimethyl-4,4′-bi-pyridilium-2A (paraquat), neomycin, dichloro-m-xyienol, and phenyl mercury acetate and chloride. Despite the many replications the high variability of the yield data does not allow of a precise classification of the relative activities. The most active compounds were 2, 4-dichloro-5-fluorophenoxyacetic acid and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T), although 2, 4-dichlorophen-oxyacetic acid (2, 4-D) gave increases in yield comparable to those of 2, 4, 5-T in some of the trials.     

Two novel genes induced by hard-surface contact of Colletotrichum gloeosporioides conidia

Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. The CHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting that C. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.