Potential for Biocontrol of Lasiodiplodia theobromae (Pat.) Griff. & Maubl. in Banana Fruits by Trichoderma Species

Fifteen Trichoderma isolates were tested for their antagonistic ability against Lasiodiplodia theobromae. Trichoderma harzianum exhibited the greatest inhibition in dual culture. Microscopic investigation demonstrated direct parasitism and coiling of T. harzianum and T. viride around hyphae of L. theobromae, causing swollen, deformed, shortened, or rounded cells of the pathogen. Granulation of cytoplasm and disintegration of the hyphal walls of L. theobromae also were noted in dual culture. Trichoderma viride reduced rotting by 29.07 to 65.06% in artificially inoculated banana fruits. Treatment of banana fruits with T. viride 4 h prior to inoculation with L. theobromae provided better protection than simultaneous application or treatment 4 h after inoculation.     

Development and characterization of microsatellite loci for the tropical tree pathogen Botryosphaeria rhodina

Nineteen simple sequence repeat (SSR) markers were developed for the pathogen and blue stain fungus Botryosphaeria rhodina. Eight pairs were found to be polymorphic among isolates collected from Pinus spp., whereas a further five pairs were polymorphic when isolates from Pinus spp. were compared with those from Eucalyptus spp. Nine isolates of B. rhodina collected from pines and eucalypts in South Africa, Mexico and Indonesia were used to demonstrate the range of the markers. Testing the markers yielded preliminary evidence that relationships among isolates are more closely linked to host than to geographical origin.     

Studies on inhibitory activities of antibodies against phytopathogenic fungi: an immunological approach

The major problem in agriculture is that of the fungal pathogens. In this era, where biological control is at focus, and is the centre of crop protection as well as environmental protection, the synthesis of new bio bodies is of utmost need. Fungicides available in the market, though of potential, are pathogen specific and highly pollutive. An attempt was made to raise polyclonal antibodies against Aspergillus niger. Following the particular standardised immunisation schedule, regular injections and periodic tapping were carried out. The IgG purified was used to check cross-reactivity with different crop fungi like Fusarium solani, Fusarium oxysporum f. sp. cicer (FOC), Rhizoctonia bataticola, Botryodiplodia theobromae, Curvularia sp., Alternaria porri and also with Aspergillus niger by two different methods. Liquid test media and the radial growth inhibition test performed in solid media were used to check the inhibition of fungi and cross-reactivity. The results were subjected to analysis of variance (ANOVA) by using Tukey's test at the significance level of p < 0.05. The antibodies were active against all the fungi for more than 15 days except for A. niger in which from the seventh day onwards spore germination was observed. The probable role of antibodies to detect the common antigenic molecule that may be present in all the tested fungi and their role in inhibiting these fungi is discussed.     

Trichoderma longibrachiatum as an antagonist of Botrydiplodia theobromae

The antagonistic potential of Trichoderma longibrachiatum against the pathogen Botrydiplodia theobromae was examined in vitro. Both fungi were paired on 9 cm Petri plates of acidified potato dextrose agar (APDA). Three pairing methods were employed, viz., ‘inoculating antagonist before pathogen’, ‘inoculating pathogen before antagonist’ and ‘simultaneous inoculation of pathogen and antagonist’. Radial growth (cm) of both fungi were later analysed using the GLM Procedure of SAS. Growth inhibition of B. theobromae by T. longibrachiatum in all pairing methods was significantly different from control (P = 0.05, R ² = 0.89). Growth inhibition of the pathogen was best when using ‘inoculation of antagonist before pathogen’. Duration of pairing (DAP) and pairing method are both critical to significant antagonistic impact of T. longibrachiatum on B. theobromae (P > 0.0001). Inhibition mechanism includes competition for space and nutrients. T. longibrachiatum could thus be a promising antagonist of B. theobromae.     

Genetic diversity and gene flow estimates among three populations of Botryodiplodia theobromae causing die-back and bark canker of pear in Punjab

Three populations of Botryodiplodia theobromae causing die-back and bark canker of pear in Punjab were analysed to know about the genetic diversity, gene flow, heterogeneity and the probable rate of spread of races capable of overcoming the resistance present in elite cultivars. There was no relationship between the morphologically similar isolates and their pathogenic behaviour. Majority of the isolates of B. theobromae within and among the populations produced lesions of 1.9–7.2 × 0.8–3.1 cm size on detached pear twigs cv. Punjab Beauty. Incubation period and per cent infection also varied within and among the three populations of the pathogen. The genetic diversity was calculated on the basis of allele frequencies of nine simple sequence repeat (SSR) markers using Nei's genetic diversity formulae. Allele frequencies (X_ij ) varied significantly among the three populations ranging from 0.0 to 1.0. The mean genetic diversities within population (H _S) also varied in all the locations ranging from 0.18 to 0.36 indicating a high genetic diversity within the population. The total genetic diversity across all the populations (H _T) ranged between 0.1 and 0.5 with a mean of 0.36, and differed significantly from the mean H _S (0.25). Diversity indices of SSR loci varied from low (H _S < 0.1) to high (H _S > 0.4) across all the populations (G _ST) of B. theobromae in Punjab which were consistent over the presumed SSR loci with a mean G _ST of 0.29. The lower G _ST values of 0.13, 0.10, 0.11 and 0.13 were observed at LAS15-16, LAS27-28, BOT17-18 and BOT35-36 loci, respectively, showing high genetic diversity among all the isolates. However, at BOT19-20 locus, the G _ST value observed was 0.72 thereby indicating low diversity in the population at this locus. Out of 25 loci, 8 loci showed the gene flow (Nm) < 1 indicating a high genetic differentiation within the population and the remaining 6 loci showed the Nm > 1 indicating the frequent existence of gene flow across the populations of B. theobromae. Pairwise comparison (G _ST) values among all the loci were ranging from 0.18 to 0.19, which indicate a low genetic differentiation among the populations of B. theobromae. The high genetic diversity (H _S) within the population was observed in the present study suggesting that B. theobromae possess high variability in Punjab. Keeping this in view, new races would be expected soon outside the state of origin, as naturally occurring gene flow is likely to be increased by the human activity for comprehensive cultivation of pear in the near future. Therefore, it is difficult to predict the durability of resistant sources in Punjab, which in turn emphasises the identification and introgression of R genes into commercial cultivars.     

Novel Potato Micro-Tuber-Inducing Compound, (3R,6S)-6-Hydroxylasiodiplodin,from a Strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10^?4 M, using the culture of single-node segments of potato stems in vitro.     

葡萄溃疡病菌限制性内切酶介导整合方法的建立及转化子库的构建

[目的]摸索葡萄溃疡病菌(Lasiodiplodia theobromae)限制性内切酶介导整合(Restriction enzyme mediated integration,REMI)的转化方法,并构建CSS-01s (L.theobromae)的REMI转化子库.[方法]利用REMI转化方法,将线性化的含有潮霉素抗性基因(Hygromycin phosphotransferase gene,Hyg)的pUCATPH质粒转化CSS-01s菌株的原生质体;测定其对潮霉素B的敏感浓度,摸索不同酶解液和酶解时间对原生质体制备的影响,统计不同限制性内切酶酶量对转化效率的影响,以摸索出的最优条件构建CSS-01s菌株的REMI转化子库,采用Southern blot的方法验证转化子.[结果]首次建立了一套L.theobromae的REMI转化方法,经转化得到包含6 000余个转化子的L.theobromaeCSS-01s转化子库,随机挑选的5个转化子经Southern blot分析,质粒均插入到了Hind Ⅲ相应的酶切位点处.[结论]浓度为2％崩溃酶+2％蜗牛酶酶混合液、酶解4h为原生质体获得的最优条件,每管转化体系中加入30 UHind Ⅲ时转化效率最高,该方法可以用来获得大量不同表型的REMI转化子.     

一种袋栽黑木耳共生菌的鉴定及其共生效应初步研究

【目的】研究袋栽黑木耳某种共生菌对黑木耳生长的影响。【方法】经分离纯化,以及形态学和分子系统学分析,鉴定该真菌的分类地位;通过不同时间接种共生菌到黑木耳菌棒的方法,研究两者的共生效应;通过子实体营养成分分析,研究共生菌对黑木耳子实体营养结构的影响。【结果】经分离纯化获得菌株B-1,形态学和分子系统学分析将该菌株鉴定为毛色二孢属的可可毛色二孢(Lasiodiplodia theobromae)。通过不同时间接种黑木耳菌棒发现,此真菌与黑木耳间存在偏性共生关系。它对于黑木耳是偏利共生,还是偏害共生取决于黑木耳菌丝在菌棒中是否具有先占优势。黑木耳子实体营养成分分析显示,可可毛色二孢的存在改变了黑木耳子实体中营养结构,但黑木耳子实体形态特征无明显变化。【结论】可可毛色二孢与袋栽黑木耳存在着偏性共生关系,该现象为首次报道。     

Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae

From the culture filtrate of the fungus Botryodiplodia theobromae five hydroxylated cyclopentane fatty acids of the jasmonic acid type were isolated and identified as (11 S -(-)-hydroxyjasmonic acid; (11R)-(-)-hydroxyjasmonic acid; (-)-12-hydroxyjasmonic acid; (-)-8ξ-hydroxyjasmonic acid; (-)-3-oxo-2-(1ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid; (-)-3-oxo-2(4ξ-hydroxy-2Z-pentenyl)cyclopent-1-yl-butyric acid. In addition, the corresponding hydroxylated iso-jasmonic acid analogues were found as minor constituents. During silica gel chromatography 11,12-didehydrojasmonic acid, 11ξ-acetoxyjasmonic acid, 3-oxo-2-(4ξ-acetoxy-2Z-pentenyl)cyclopent-1-yl-butyric acid 3-oxo-2-(2Z,4-pentadienyl)cyclopent-1-yl-butyric acid were formed as artefacts.