Reclassification of an isolate of Guignardia citricarpa from New Zealand as Guignardia mangiferae by sequence analysis

Citrus black spot was recorded as present in New Zealand in international databases on the basis of one isolate (ICMP 8336) identified by morphological features as Guignardia citricarpa . This isolate was from a soft rot, not a typical symptom of citrus black spot, on a Seminole tangerine fruit grown in the northern region of New Zealand. Sequence analysis of the internal transcribed spacer (ITS) region (ITS1, 5·8S and ITS2) showed that this isolate was 99% identical to the ITS region of G. mangiferae , a closely related saprotroph. Despite climatic conditions being suitable for this disease, citrus black spot symptoms have never been seen on citrus fruits grown in New Zealand. Thus the absence of symptoms on citrus in New Zealand is probably because G. citricarpa is not present. On the basis of these results, the record of citrus black spot occurring in New Zealand should be re-examined.     

Spatial pattern of black spot incidence within citrus trees related to disease severity and pathogen dispersal

Guignardia citricarpa , the causal agent of citrus black spot, forms airborne ascospores on decomposing citrus leaves and water-spread conidia on fruits, leaves and twigs. The spatial pattern of diseased fruit in citrus tree canopies was used to assess the importance of ascospores and conidia in citrus black spot epidemics in São Paulo State, Brazil. The aggregation of diseased fruit in the citrus tree canopy was quantified by the binomial dispersion index ( D ) and the binary form of Taylor's Power Law for 303 trees in six groves. D was significantly greater than 1 in 251 trees. The intercept of the regression line of Taylor's Power Law was significantly greater than 0 and the slope was not different from 1, implying that diseased fruit was aggregated in the canopy independent of disease incidence. Disease incidence ( p ) and severity ( S ) were assessed in 2875 citrus trees. The incidence-severity relationship was described ( R ² = 88·7%) by the model ln( S ) = ln( a ) +  b CLL( p ) where CLL = complementary log-log transformation. The high severity at low incidence observed in many cases is also indicative of low distance spread of G. citricarpa spores. For the same level of disease incidence, some trees had most of the diseased fruit with many lesions and high disease severity, whereas other trees had most of the fruit with few lesions and low disease severity. Aggregation of diseased fruit in the trees suggests that splash-dispersed conidia have an important role in increasing the disease in citrus trees in Brazil.     

Development and Validation of a Fast PCR-Based Detection Method for Pathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa

Based on the ITS regions of the ribosomal DNA, specific primer sets were developed for the citrus pathogen Guignardia citricarpa and the common citrus endophyte, G. mangiferae, and tested for their specificity against 37 isolates of G. citricarpa, 29 isolates of G. mangiferae, 10 isolates of related species and other fungi found on citrus. The efficacy of the PCR-detection method for G. citricarpa was approximately 60–70% for lesions without pycnidia, and approximately 90% for lesions with pycnidia. A reliability of 99% can be reached by analysing multiple lesions per sample. An internal control was developed to monitor DNA samples for PCR inhibition; samples with PCR inhibition should be re-examined. Detection by PCR is more rapid than the current five-day incubation method prescribed by the European Union for diagnosis of black spot lesions lacking the diagnostic pycnidia. The latter method had an efficacy of 40–50%, while culturing of suspected lesions had an efficacy of 10%. Species-specific primers and ITS sequence data showed that G. citricarpa can occur as a symptomless endophyte in leaves. This shows that wild and cultivated plants occurring in citrus groves are potential carriers of this quarantine fungus. Application of the presently developed PCR method for the detection of G. citricarpa will enable citrus producing as well as importing countries to prevent further spread of this harmful organism.     

Crop loss, aetiology, and epidemiology of citrus black spot in Ghana

Citrus Black Spot (CBS), caused by Guignardia citricarpa, was detected for the first time in Ghana and in West Africa. The disease was first observed in the Eastern Region in 1999 with typical disease symptoms including hard spot, virulent spot and false melanose were observed on several citrus species. A survey revealed that the disease has reached epidemic levels in the citrus-producing areas of the Eastern and Ashanti regions and is spreading rapidly within these areas and to other regions of the country. Currently, CBS is the most important fruit disease of citrus in Ghana, causing about 22% crop loss. Although the disease does not cause postharvest decay and the internal quality of the fruit is not affected, significant amounts of blemished fruit are discarded at the markets. Disease incidence and severity was found to be higher on mature than on young citrus trees. Pycnidia were found on fruit with hard spot symptoms, and pycnidia and pseudothecia typical of Guignardia spp. were found on decomposing leaves. Two species, G. citricarpa and G. mangiferae, were isolated from 15% of the samples collected and identified using the Oatmeal Agar test and by PCR with species-specific DNA primers. Isolates of G. citricarpa produced CBS symptoms after 80 to 233?days on 75% of the artificially inoculated young fruit of Valencia Late sweet orange. The fungus was re-isolated from symptomatic, inoculated fruit completing Koch’s postulates. Isolates of the endophyte G. mangiferae did not induce symptoms in the pathogenicity tests. In epidemiological studies, infections were detected from November to February for the minor cropping season and from May to November for the major season. Fruit of Valencia Late sweet orange were susceptible to G. citricarpa infection for up to 7?months after petal fall. Knowledge of the disease cycle in Ghana will improve methods for disease control.     

Leptosphaeria nodorum on wheat in Ireland

Symptomless leaves of young wheat crops collected in January and February produced, after surface-sterilization and incubation on an agar medium, perithecia typical of Leptosphaeria nodorum. Single-ascospore cultures produced pycnidia typical of Septoria nodorum, whose conidia caused necrotic lesions to develop when sprayed on wheat seedlings.     

Fruit spot of sweet lime (<Emphasis Type="Italic">Citrus limetta</Emphasis>) caused by <Emphasis Type="Italic">Septoria</Emphasis> sp. in Peru

In 2002, a severe fruit spot of sweet lime (Citrus limetta) was observed in Piura and Lambayeque provinces in northern Peru. Affected fruits showed large oval and sunken lesions, often surrounded by chlorotic haloes. Septoria sp. was isolated from affected fruits. Sweet lime isolates showed larger pycnidia and pycnidiospores than those of Septoria spp. previously described on citrus. In addition, phylogenetic analysis of the ITS sequences clearly separated the sweet lime isolates from S. citri and S. citricola. Isolates were pathogenic to detached sweet lime fruits and the fungus was isolated from lesions on inoculated fruits.     

Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil

Brazilian isolates of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop were examined for colony colour, mycelial growth, benomyl-resistance, pathogenicity, and genetic variability by random amplified polymorphic DNA (RAPD) analysis. All isolates were obtained from flowers and persistent calyxes from different citrus hosts from Sao Paulo, Brazil. DNA polymorphisms detected after amplification with random 10-mer primers were used to classify the isolates into two groups. Group I isolates grew rapidly on potato-dextrose agar (PDA) and were sensitive to benomyl, and group II isolates grew slowly on PDA and were benomyl-resistant. Colletotrichum acutatum was analyzed by RAPD and had high genetic similarity with group II isolates of Colletotrichum from citrus. Probably, the group I is C. gloeosporioides and group II is C. acutatum.     

苹果炭疽叶枯病病原Glomerella cingulata及其侵染过程

为明确苹果炭疽叶枯病病原菌围小丛壳Glomerella cingulata的侵染致病特征,在分离获得该病原菌的基础上,采用形态学观察、ITS序列分析和致病性测定对其进行了鉴定,并利用光学和扫描电子显微镜对病原菌在嘎啦苹果叶片上的侵染过程进行了研究.结果表明,在陕西咸阳地区分离获得的9株病原菌均为围小丛壳G.cingulata.25 ℃下接种9 h后,分生孢子中间产生隔膜,双胞化,并萌发产生芽管和附着胞;24 h后分生孢子的2个细胞均可萌发并形成芽管及附着胞,部分芽管顶端可产生次级分生孢子;48 h后次级分生孢子萌发形成附着胞;72 h后,附着胞下形成的侵染钉可直接入侵寄主,在表皮细胞内形成初生菌丝和次生菌丝,此时叶片表面已出现褐色斑点.接种7 d后叶片病斑处出现分生孢子盘和子囊壳.表明陕西省近年出现的苹果炭疽叶枯病病原菌为围小丛壳G.cingulata,该病菌在嘎啦叶片上的一些特殊侵染行为可能是导致该病害易在短时间内暴发的重要原因.     

Characterization of Botryosphaeria dothidea Isolates Collected from Pistachio and Other Plant Hosts in California

ABSTRACT Eighty-six isolates of Botryosphaeria dothidea, the causal agent of Botryosphaeria panicle and shoot blight of pistachio, were collected from pistachio and other plant hosts in California. The isolates were characterized by microsatellite-primed polymerase chain reaction (MP-PCR), sequences of the nuclear ribosomal DNA internal transcribed spacer region (ITS1, 5.8S gene, and ITS2), morphological and cultural characters, osmotic and fungicide sensitivity, and pathogenicity on pistachio. Three groups of these isolates were identified based upon analysis of MP-PCR data and ITS sequences. Group I contained 43 pycnidiospore-derived isolates collected from pistachio and other hosts. Group II consisted of 20 ascosporic isolates obtained from a single sequoia plant. Group III consisted of 20 ascosporic isolates from three shoots on a single blackberry plant, two pycnidiospore-derived isolates from incense cedar, and one from pistachio. Group I predominated over the other two groups in California pistachio orchards. B. dothidea isolates of group III grew faster on acidified potato dextrose agar (APDA) than the isolates of groups I and II. Isolates of group III produced pycnidia on both APDA and autoclaved pistachio shoots, but the isolates of the other two groups produced pycnidia on only autoclaved pistachio shoots. Additionally, significant differences in osmotic and fungicide sensitivities were observed among these three groups. Results from lathhouse inoculations demonstrated that the representative isolates for each of the three groups were all capable of infecting pistachio and producing characteristic disease symptoms of Botryosphaeria blight. The virulence of group II isolates on pistachio was, however, significantly lower than that of group I isolates.     

柑橘叶点霉属(Phyllosticta spp.)真菌的遗传多样性分析

利用筛选的11条ISSR引物对从我国柑橘主产区和国外收集的135株叶点霉属真菌菌株进行扩增,扩增产物进行凝胶电泳,扩增图谱用NTSYS-pc2.10软件进行群体聚类分析。结果显示,共扩增出116个DNA条带,其中多态性位点为112个,多态率为96.55%;平均每个引物可以扩增出10.55个条带,扩增产物大小在250~3 000 bp。聚类分析显示,中国柑橘叶点霉属真菌可以分为4个种,即P.citricarpa、P.citriasiana、P.capitalensis和P.citrichinaensis。以遗传相似性系数0.97为阈值时,柑橘黑斑病病原菌(P.citricarpa)种群可分为5个亚类,subclade-I的菌株来自中国的本地早、温州蜜柑、槾橘和南丰蜜橘,subclade-II的菌株均来自中国的砂糖橘,subclade-III的菌株来自非洲莫桑比克柠檬和葡萄柚、佛罗里达甜橙、南非甜橙和来自杭州市场上进口的澳橘,subclade-IV的菌株来自中国的甜橙,subclade-V的菌株来自中国的柠檬,表明我国柑橘黑斑病病原菌具有丰富的遗传变异,其遗传变异与寄主相关;我国与国外的柑橘黑斑病病原菌在起源上可能存在差异。P.citriasiana、P.capitalensis和P.citrichinaensis种群内也存在遗传变异,但其遗传变异与地理分布和寄主未发现相关性。该研究结果为研究柑橘叶点霉属真菌的遗传多样性奠定了基础。     

一种新的香蕉黑斑病病原菌鉴定及生物学特性研究

 在广西香蕉种植区发现一种引起香蕉黑斑病的新病原。通过对病原菌分离培养、致病性测定、形态学观察、分子生物学鉴定及对该病原菌生物学特性进行初步研究,表明引起该病害的病原菌为芒果球座菌(Guignardia mangiferae A.J. Roy)。该病原菌菌落为深橄榄色,产生棒状子囊、梭形子囊孢子及倒梨形分生孢子;其最适生长温度为28℃,最适pH 值为6;病原菌生长较好的碳源为果糖和阿拉伯糖,氮源为蛋白胨和酵母膏;在全光条件下,病原菌菌丝扩展速度最快;菌丝的致死温度为54℃。由G. mangiferae引起的香蕉黑斑病,在国内外为首次报道。     

Toxin Profile, Fertility and AFLP Analysis of Fusarium verticillioides from Banana Fruits

Gibberella fujikuroi is composed of at least nine mating populations (MPs), corresponding to biological species and assigned letters (from A to I). Each MP possesses a specific toxicological profile and a preferential host. Members of Fusarium verticillioides and F. thapsinum, anamorphs respectively of MPs A (G. moniliformis) and F (G. thapsina), share identical morphological traits, but they have a different preferential hosts (maize and sorghum, respectively) and toxin profiles, beingable the only member of MP A to produce fumonisins and the only member of MP F to produce moniliformin. Isolates from banana fruits were identified morphologically as F. verticillioides. The isolates were analyzed for fumonisin and moniliformin production. While none of the isolates produced fumonisin, all the isolates produced moniliformin. The isolates were crossed with tester strains of MPs A and F, showing ability to produce fertile perithecia only when crossed by MP A tester strains isolated from maize. However, the time required for the formation of fertile perithecia and their size differed significantly from the usual fertile crosses of strains belonging to MP A. Pathogenicity tests using such isolates of F. verticillioides isolated from banana and a set of F. verticillioides isolates isolated from maize were also performed on banana fruits. The data showed that the isolates from banana were more pathogenic. Finally, isolates from banana and maize were compared using AFLP. The results revealed that isolates from banana and maize produced two distinctly different clusters. In conclusion, isolates of F. verticillioides from banana showed specific traits (toxin production, in vitro fertility, pathogenicity and molecular profiles), that were different to those of the same species from maize. This could reflect important differences in the ecology and natural history of the population from banana and should encourage further investigations into the mechanisms of toxin production and pathogenicity within the same MP.     

漳州琯溪蜜柚果实黑斑病菌的分子鉴定

2008年从漳州采集到疑似由柑橘球座菌(Guignardia citricarpaKiely)引起的黑斑病症状的溪琯蜜柚(Citrus grandis(Linn.)Osbeck cv.guanxi-miyou)果实。使用柑橘球座菌(G.citricarpa)特异引物对该果实上的病斑进行PCR检测及序列分析。PCR检测结果表明从溪琯蜜柚果实病斑上扩增到约490bp的条带。序列分析结果表明,与已知的亚洲柑橘叶点霉(Phyllosticta citriasiana)、G.citricarpa和G.mangif-erae的相应序列同源性分别为99.8%、98.2%和94.0%。该序列含有部分的内转录间隔区1(ITS1)、5.8S核糖体RNA基因(5.8S rRNA)和部分的内转录间隔区2(ITS2)序列。结果表明,引起溪琯蜜柚果实病害的病原菌并不是G.citricarpa,而是一种新描述的病原菌——亚洲柑橘叶点霉(Phyllosticta citriasianaWulan-dari,Crous&Gruyter),该病原菌引起的病害称为柑橘褐斑病。     

Phylogenetic analysis based on ITS sequences and conditions affecting the type of conidial germination of Bipolaris oryzae

One taxonomic characteristic of Bipolaris species is the bipolar germination of conidia, but conidia of Bipolaris oryzae , the causal pathogen of brown spot in rice, are regularly observed to show intercalary germination, a characteristic of Drechslera species. The effect of selection, culture media and culture age on type of conidial germination was determined for three brown spot isolates from Cavinti, San Pablo and Palawan in the Philippines, obtained from infected leaves showing typical disease symptoms. Based on the analyses of their ITS1, ITS2 and 5·8S rDNA nucleotide sequences, the local isolates were clearly identified as B. oryzae . Selection for colonies of the three isolates derived from single conidia with either bipolar or intercalary germination had no effect on the number of spores showing bipolar germination in subsequent cultures. Germination on seven different culture media was tested; of these, rabbit food agar and water agar increased the percentage of bipolar germination of conidia, although this varied between isolates. Incubation of the cultures of all three isolates for longer periods prior to harvesting conidia increased the percentage of bipolar-germinating conidia from c . 40 to c . 90% with 5-day-old and 30-day-old cultures, respectively.     

温度、湿度和光照对苹果炭疽叶枯病菌(Glomerella cingulata)产孢的影响

苹果炭疽叶枯病(Glomerella cingulata)是我国苹果上新发现的一种病害,为了了解病原菌的产孢条件和产孢动态,为病害的预测预报与防控提供依据。本研究在人工控制条件下,测试了温度、湿度和光照对苹果炭疽叶枯病菌产生分生孢子和子囊孢子的影响。结果表明,苹果炭疽叶枯病新形成的病叶润湿后,在15℃~30℃下保湿培养2~6 d后可产生大量橘黄色的分生孢子堆,其中30℃下产孢量最大,产孢速度最快,仅需2 d时间。炭疽叶枯病菌在新形成的病叶上于15℃~30℃下培养20~30 d可形成子囊孢子,最适温度为25℃,子囊孢子的形成需要高湿环境或叶片润湿。炭疽叶枯病菌的单孢分离菌株于15℃~25℃下,在马铃薯葡萄糖琼脂培养基(PDA)上培养20~30 d也可形成子囊孢子,最适产孢温度25℃。紫外光、黑光和日光都能促进子囊孢子的形成。     

福建省柑橘褐斑病菌的分离、鉴定和系统进化分析

为明确在福建省南平市的橘柚和三明市的温州蜜橘上发现的疑似柑橘褐斑病的病原菌种类,采用组织分离法获得纯化菌株,通过回接法验证菌株的致病性,利用形态学特征对病原菌进行初步鉴定,并采用最大似然法以多聚半乳糖醛酸酶基因endoPG为靶标对本研究以及国内外已报道的链格孢菌株构建系统发育树,分析其遗传多样性。结果表明,从病组织中共分离获得26株纯培养菌株,经形态学鉴定均为链格孢菌Alternaria spp.。利用分生孢子液接种橘柚离体嫩叶发现,有22株菌株能侵染橘柚叶片并产生与田间相似的褐斑病症状,确认该病害为链格孢引起的柑橘褐斑病。系统发育树分析结果显示,分离所得的26株菌株均聚在已报道的4个柑橘链格孢进化分支Clade1～Clade4中,其中21株菌株聚在国内特有的分支Clade4中,有3株菌株和1株菌株分别聚在国内外兼有的分支Clade3和Clade1中,1株菌株聚在国外特有的分支Clade2中,表明在福建省采集的这些柑橘褐斑病菌均为链格孢菌,且遗传多样性较丰富。     

Potential distribution of citrus black spot in the United States based on climatic conditions

Citrus black spot (CBS), caused by Phyllosticta citricarpa McAlp Van der Aa, was recently detected in southern Florida in the US. In addition to infected plant propagation materials, movement of infected citrus fruit poses a concern for potential spread of the disease out of the current quarantine zone, because lesions with pycnidia and conidia could develop after harvest. The conditions conducive for mycelial growth and development of pycnidia and conidia are not well known. Therefore, effects of temperature and relative humidity on growth and conidial production of P. citricarpa were determined and used as parameter inputs in CLIMEX to predict potential establishment of CBS in North America. Colony growth and conidial production in vitro were optimal at 27 °C, whereas there was no growth below 4 °C and above 37 °C. On fruit, lesion development and conidial production were observed at 4 °C, though at a low rate, indicating a greater versatility of the fungus on fruit. More full pycnidia were produced on the CBS lesions at 91 % RH compared to 84 %. Input parameters for CBS risk in CLIMEX obtained from literature, which reflected conditions for infection in spring/summer in Florida, predicted potential establishment in Florida but not in California. However, altering the parameter values to account for survival of the pathogen in leaf litter in winter predicted potential establishment in California as well as Florida. Thus, P. citricarpa could possibly establish beyond Florida if this organism is transported outside of the current quarantine zone to other citrus production areas.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

中国柑橘黑腐病和褐斑病病原菌的系统发育分析

 近些年链格孢属真菌的分类有了长足的进展,但柑橘褐斑病和黑腐病的病原却还是存在着一些争议。本研究从中国柑橘果实上收集了49株黑腐病菌菌株,并选取了8株从柑橘叶片上分离的具代表性的褐斑病菌菌株,基于Alta1 、endoPG、LSU、OPA10-2、OPAl-3和OPA2-1 等6个基因位点串联序列构建系统发育树。结果表明,柑橘黑腐病和褐斑病都可由不止一种链格孢菌引起,均以Alternaria alternata为主。两种病害的病原菌之间不能通过该系统发育树区分,但在致病性上存在差异,且能通过扩增ACT毒素合成基因进行区分。为了使两种病害的病原更加方便阐述,作者建议以它们的主要类群对其进行命名,柑橘褐斑病病原学名还是遵循前人的称呼,为the tangerine pathotype of A. alternata,即链格孢菌橘致病型,而柑橘黑腐病病原应为A. alternata,即链格孢菌。