Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis)

ABSTRACT The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa. This distinction was supported by amplified fragment length polymorphism analysis that also supported the exclusion of two isolates that had apparently been misclassified as G. citricarpa. On cherry decoction agar, but not on other media, growth rates of group I isolates were lower than those of group II isolates. Conidial dimensions were similar, but group I isolates formed conidia with barely visible mucoid sheaths, whereas those of group II formed conidia with thick sheaths. Cultures of isolates belonging to group I produced rare infertile perithecia, whereas fertile perithecia were formed by most isolates of group II. Colonies of isolates belonging to group I were less dark than those of group II, with a wider translucent outer zone and a lobate rather than entire margin. On oatmeal agar, exclusively group I isolates formed a yellow pigment. Group I harbored strains from citrus fruits with classical black spot lesions (1 to 10 mm in diameter) usually containing pycnidia. Group II harbored endophytic strains from a wide range of host species, as well as strains from symptomless citrus fruits or fruits with minute spots (<2-mm diameter) without pycnidia. These observations support the historic distinction between slowly growing pathogenic isolates and morphologically similar fast-growing, nonpathogenic isolates of G. citricarpa. The latter proved to belong to G. mangiferae (P. capitalensis), a ubiquitous endophyte of woody plants with numerous probable synonyms including G. endophyllicola, G. psidii, P. anacardiacearum, and P. theacearum. G. mangiferae occurs in the European Union and the United States on many host species including citrus, and does not cause symptoms of citrus black spot, justifying its exclusion from quarantine measures.     

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。     

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.     

A new method for inoculation of fruit with <Emphasis Type="Italic">Guignardia citricarpa</Emphasis>, the causal agent of citrus black spot

Citrus black spot (CBS) is a fungal disease, caused by Guignardia citricarpa, that has a high economic impact on citrus. Although G. citricarpa has been associated with black spot of citrus, an adequate pathogenicity test is still not available. Thus, our objective was to develop and evaluate a simple, safe, and practical pathogenicity test. We used fruits from Pera-Rio and Valencia sweet orange trees from two different orchards, located in the State of São Paulo, Brazil. Inoculation was performed by placing six disks colonized by G. citricarpa, onto the peel of healthy fruits, previously bagged. In the Pera-Rio sweet orange grove, initial symptoms of the false melanose type resulting from the inoculations were observed 55 days after inoculation (dai). In the Valencia grove, initial symptoms also of the false melanose type resulting from the inoculations occurred 73 dai. A total of 92.8% and 86.6% of the Pera Rio and Valencia fruits inoculated, respectively, showed symptoms of CBS. Citrus black spot symptoms were not observed in any of the control fruits.     

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.     

Sooty spot caused by Cladosporium cladosporioides in postharvest Satsuma mandarin grown in heated greenhouses

In June 2006, a large number of small black spots containing a branching network of black hyphae developed on shipped Satsuma mandarin fruits grown in greenhouses, which markedly decreased their commercial value. The fungus that was frequently isolated from the damaged fruits was identified as Cladosporium cladosporioides. Inoculation of fruits with the fungus caused the same symptoms, and the fungus was reisolated from the diseased fruits. The disease caused by this fungus has not been previously documented in citrus fruits; we name this new disease “sooty spot” on the basis of its symptoms.     

几种药剂防治柑橘黑斑病的田间药效试验

为筛选出防治柑橘黑星病的有效药剂,以南丰蜜橘为研究对象,对柑橘黑斑病进行田间防治试验。结果表明:10%苯醚甲环唑1000倍防治效果最好,防治效果为92.32%;80%代森锰锌+70%甲基托布津复配500倍防治效果次之,防治效果为91.45%;依次是80%代森锰锌400倍、70%甲基托布津400倍和10%苯醚甲环唑1500倍,防治效果分别为87.32%、84.65%和83.56%。     

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.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

广东梅州蜜柚柑橘黑斑病菌的检验鉴定

近年,柑橘黑斑病成为阻碍我国柑橘鲜果出口贸易的瓶颈,广东梅州名优鲜果蜜柚生产基地也遇到类似情况。该病发生后,病果果皮表面产生许多圆形病斑,初呈红褐色,中间凹陷,直径多为2~3mm,后期病斑逐渐转为褐色至黑褐色,病斑中部明显可见细小的黑色粒点状分生孢子器。病果分离物的培养性状与形态特征与柑橘黑斑病菌(柑橘球座菌,Guignarida citricarpa)一致。采用真菌ITS通用引物和柑橘球座菌特异性引物,从病斑组织基因组DNA及分离物基因组DNA中分别扩增得到541bp和396bp的片段。BLAST分析表明:扩增到的片段均与GenBank中已知柑橘球座菌菌株的rDNAITS序列相似性最高,分别为97%和98%。根据分离菌株的培养性状和形态特征及ITS区序列分析结果,将梅州蜜柚上的柑橘黑斑病菌鉴定为柑橘球座菌(Guignarida citricarpa Kiely)。     

来源于猕猴桃的柑橘叶斑驳病毒的RT-PCR检测及外壳蛋白基因序列分析

柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)为柑橘病毒属(Citrivirus)代表种。本研究采用RT-PCR技术从我国栽培猕猴桃上首次检测到CLBV,总检出率为13.3%。测定了5个CLBV分离物的外壳蛋白基因(coat protein gene,cp)序列,全长均为1 092核苷酸(nt),分离物间cp基因核苷酸和编码氨基酸序列相似性分别为83.2%~99.7%和91.9%~98.9%,这些分离物与新西兰报道的CLBV猕猴桃分离物M3-A核苷酸序列相似性仅为83%左右,与来源于柑橘及近缘种的CLBV分离物cp基因核苷酸序列相似性为84%~86%。在基于该病毒cp基因核苷酸序列的系统进化树中,本研究所测定的CLBV猕猴桃分离物与新西兰报道的除M3-A外的猕猴桃分离物聚在同一组群。研究结果为进一步明确CLBV在我国猕猴桃上的侵染和危害特点及建立该病毒的分子检测技术提供了重要信息。     

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.     

Histopathology of black spot symptoms in sweet orange<Emphasis Type="Italic">s</Emphasis>

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.     

Epidemiological aspects of Phyllosticta citricarpa colonization and viability in Citrus sinensis

Journal of Plant Diseases and Protection - Citrus black spot (CBS) is a disease caused by the fungus Phyllosticta citricarpa that is associated with significant yield losses. The pathogen forms...     

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。     

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。     

云南蓝莓叶斑病的病原菌鉴定

<正>蓝莓(Vaccinium spp.)属于杜鹃花科(Ericaceae)越橘属(Vaccinium)多年生小浆果类果树,广泛应用于医药、保健、化妆品和环境保护等领域,联合国粮农组织将其果实列为世界五大健康食品之一。我国已有10多个省份开始大面积的蓝莓商业化栽培[1],这也是南方酸性土丘陵地区值得发展的经济作物。随着栽培面积的不断扩大,蓝莓病害日趋突出,严重影响和制约蓝莓产业的发展。     

Genetic variability and pathogenicity of Rhizoctonia solani associated with black scurf of potato in New Zealand

Isolates (a total of 129) of Rhizoctonia solani were collected from black scurf on potato tubers from different potato‐growing regions in New Zealand. Sequence analysis of the nuclear ribosomal DNA internal transcribed spacer (rDNA–ITS) regions from these isolates identified three anastomosis groups (AGs), AG‐3PT, AG‐2‐1 and AG‐5. Isolates classified as AG‐3PT were widely distributed, whereas AG‐2‐1 and AG‐5 were confined to distinct locations. Sequence heterogeneity was identified in the ITS regions of 100 AG‐3PT and AG‐2‐1 isolates. Variation in the sequence and length of the rDNA–IGS1 region was also observed for selected isolates of AG‐3PT and AG‐2‐1. Phylogenetic studies found all AG‐2‐1 isolates belong to AG‐2Nt, a subset of AG‐2‐1 previously associated with solanaceous crops in other countries. AG‐2‐1 isolates were consistently more aggressive than those of AG‐3PT. Delayed emergence, severe infection on stolons, formation of aerial tubers and considerable yield losses were associated with AG‐2‐1, but they caused negligible black scurf. In contrast, AG‐3PT caused black scurf on progeny tubers but variable effects on stem emergence and stolons. Furthermore, AG‐2‐1 isolates caused severe tuber malformation, but isolates of other AGs did not. This is the first report on the AG composition, genetic variability and pathogenicity of R. solani isolates associated with black scurf of New Zealand potatoes.     

新病害—山核桃果实黑斑病病原菌的鉴定

2007年以来,浙江临安、淳安、桐庐和安徽部分地区山核桃主产区出现了一种果实新病害——山核桃果实黑斑病,该病严重影响山核桃果实的产量和品质。本研究从采集的病果中分离得到了一种真菌,离体幼果接种的症状与田间相似。病原菌在PDA培养基上形成白色轮纹状菌落,分生孢子长梭形,直或弯曲,(22.6～29.9)μm×(6.1～9.0)μm,5个细胞,中间3个有色孢(上部2个为黑褐色、下部1个为淡橄榄色),孢子顶生2～3根附属丝、多为3根。根据该病原菌的培养和形态特征及其ITS序列,将其鉴定为小孢拟盘多毛孢(Pestalotiopsis microspora)。