Characterization and pathogenicity of Pestalotiopsis uvicola causing black leaf spot on carob (Ceratonia siliqua L.) in Italy

During spring 2013, several carob plants (Ceratonia siliqua L.), showing symptoms of necrotic black lesions on leaves, were observed in Capri Island, Southern Italy. Acervuli, producing conidia with septa and appendages, appeared in the centre of the lesions. In this report, the identification of the causal agent of this disease was made applying Koch’s postulates. Morphological characteristics of colony (colour and mycelium appearance) and conidia (size, shape, number of septa, length and number of apical and basal appendages) were characterized to identify the pathogen. Moreover, for a rapid and unambiguous identification of the fungal species, the internal transcribed spacers, comprising the 5.8 rDNA gene (ITS1-5.8-ITS2), were amplified by PCR, using the genomic DNA extracted from the isolated colonies, and sequenced. The fungus isolated was ascribed to the species Pestalotiopsis uvicola (Speg.) Bissett. Phylogenetic analysis, performed with sequences present in GenBank database, showed that carob isolates clustered with P. uvicola isolates from North America and Italy, separately from Australian, Indian and Chinese isolates. P. uvicola isolates were pathogenic when artificially inoculated on carob leaves. To the best of our knowledge, this is the first report of P. uvicola on carob.     

Characterisation of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> isolates from <Emphasis Type="Italic">Vitis vinifera</Emphasis> and potential biocontrol activity for the management of bitter rot of grapes

Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.     

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.     

Identification and characterization of Pestalotiopsis spp. causing twig blight disease of bayberry (Myrica rubra Sieb. & Zucc) in China

Bayberry (Myrica rubra) is a fruit tree native to the subtropical regions of China. It produces fruit with a unique taste and pharmacological characteristics, thus making it a widely cultivated tree commercially in many regions of China, including Zhejiang, Fujian, Guizhou, Sichuan and Yunnan Provinces. Recently, a twig blight disease occurred on the fruit tree and caused destructive damage of plantings in the Zhejiang Province. However, the etiology of the disease was unclear. This study was carried out to identify the causal agent(s) of the blight disease on bayberry. Fungal isolates were obtained from blighted twig samples collected from bayberry fields in Xianju, Rui’an, and Huangyan of Zhejiang Province. The majority (87.9 %) of the 257 fungal isolates were identified as Pestalotiopsis spp. based on their conidial morphology. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene and the β-tubulin gene were obtained from six representative strains (XJ27, XJ42, RA2-1, YS26, YS44 and RA1-2) of the Pestalotiopsis spp. Phylogenetic analysis showed that three of the strains (XJ27, XJ42, and RA2-1) grouped with P. versicolor (Speg.) Steyaert while the other three strains (YS26, YS44 and RA1-2) grouped with P. microspora (Speg.) Batista & Peres. Pathogenicity tests in the greenhouse showed that all these six isolates of Pestalotiopsis spp. caused twig blight disease symptoms on bayberry plants, which were the same as observed in naturally infected plants in the field. Our results clearly indicated that P. versicolor and P. microspora were the major pathogens causing the twig blight disease on bayberry in southern China.     

First report of leaf spot disease on oil palm caused by Pestalotiopsis theae in Thailand

A leaf spot on oil palm, caused by Pestalotiopsis theae, was found in a plantation of Elaeis guineensis for the first time in the world in Chiang Mai Province, Thailand. The fungus was isolated from lesions on leaves, and its pathogenicity was confirmed. Pathogenicity tests showed that P. theae could infect E. guineensis, which developed the same symptoms after inoculation as those observed naturally in the field. The fungus was identified based on morphological characteristics and confirmed using comparisons of DNA sequences of internal transcribed spacer (ITS)1, ITS2 and 5.8S rDNA. This report is the first on oil palm leaf spot disease caused by P. theae.     

Phylogenetic relationships,pathogenicity and fungicide sensitivity of Greeneria uvicola isolates from Vitis vinifera and Muscadinia rotundifolia grapevines

Greeneria uvicola causes bitter rot on Vitis vinifera (bunch grapes) and Muscadinia rotundifolia (muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship of G. uvicola representatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–5?8S–ITS2), 28S large subunit (LSU) nuclear rDNA and β‐tubulin‐2. Greeneria uvicola isolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates from V. vinifera; USA isolates from M. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic on V. vinifera (cv. Chardonnay) berries although those originating from M. rotundifolia were not as aggressive as isolates from V. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.     

Identification and species status of the mango biotype of Colletotrichum gloeosporioides in Ghana

Research work was carried out to identify and ascertain the species status of the mango biotype of Colletotrichum gloeosporioides infecting mangoes in Ghana. Forty five isolates of Colletotrichum species were collected from 12 districts in Ghana while five each were obtained from mango fruits from Florida, Mexico and Puerto Rico. The entire internal transcribed spacer region, partial beta-tubulin gene and partial glyceraldehyde-3-phosphate dehydrogenase gene of isolates were sequenced and used in phylogenetic studies. The results of the sequence analysis of the first ribosomal transcribed spacer (ITS 1) region showed that 35 % of the isolates from Ghana and all the five isolates from Mexico were the mango biotype of C. gloeosporioides, while the others were not. Phylogenetic studies showed that the mango biotype of the pathogen was Colletotrichum asianum but not C. gloeosporioides as previously thought. However, the other isolates that were not the mango biotype were identified as Colletotrichum siamense and Colletotrichum species which had probably cross-infected mango from other fruit crops in the field.     

Characterisation of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis> by RAPD-PCR,microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for <Emphasis Type="Italic">in planta</Emphasis> PCR detection

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.     

Characterization of colletotrichum isolates from tamarillo, passiflora, and mango in Colombia and identification of a unique species from the genus

ABSTRACT This study was conducted to identify the species of Colletotrichum infecting tamarillo, mango, and passiflora in Colombia and to assess whether cross-infection between host species is occurring. Isolates of Colletotrichum spp. from tamarillo (n = 54), passiflora (n = 26), and mango (n = 15) were characterized by various molecular methods and by morphological criteria. Morphological characterization grouped the tamarillo isolates as C. acutatum and the passiflora and mango isolates as C. gloeosporioides. Species-specific primer analysis was reliable and confirmed grouping of the tamarillo isolates (besides Tom-6) as C. acutatum and the mango isolates (besides Man-76) as C. gloeosporioides. However, DNA of the passiflora isolates was not amplified by either C. acutatum- or C. gloeosporioides-specific primers, but reacted with a new primer, Col1, designed according to the internal transcribed spacer (ITS) 1 region of these isolates. Isolates Tom-6 and Man-76 also reacted positively with the Col1 primer. All the isolates reacting with the C. acutatum- and C. gloeosporioides-specific primers failed to react with primer Col1. Isolate Pass-35 from passiflora did not react with any of the taxon-specific primers. Arbitrarily primed polymerase chain reaction (ap-PCR), random amplified polymerase DNA (RAPD)-PCR, and A+T-rich DNA analyses delineated representative isolates into subgroups within the designated species. Molecular analyses indicated that the C. acutatum tamarillo isolates were uniform or clonal, whereas the C. gloeosporioides mango isolates and Colletotrichum passiflora isolates were heterogeneous. Likewise, sequence analysis of the complete ITS (ITS1-5.8S-ITS2) region identified certain isolates to their respective species: tamarillo isolates as C. acutatum; mango isolates as C. gloeosporioides; passiflora, Tom-6, and Man-76 isolates as a Colletotrichum sp. as yet undefined; and the Pass-35 isolate as an additional undefined Colletot-richum sp. Molecular analyses of the population of Colletotrichum isolates from passiflora, Tom-6 from tamarillo, and Man-76 from mango indicate that this population may not be host specific.     

Biological characterization and genetic diversity analysis of two species of Pestalotiopsis causing twig dieback of Myrica rubra

Chinese bayberry (Myrica rubra Siebold & Zucc) is an evergreen fruit tree with high ecological and economic values in China. In recent years, a new twig dieback disease caused by Pestalotiopsis mangiferae and P. vismiae was observed in major M. rubra-producing areas of Zhejiang and Fujian Provinces, causing serious economic losses. In this study, 16 isolates of P. mangiferae and 27 isolates of P. vismiae were obtained from diseased leaves, roots and branches of M. rubra from different regions. The optimum growth temperature of the two species of Pestalotiopsis was determined to be 20–25 °C, while the optimum temperature for the germination of conidia was 25–35 °C. The two species of Pestalotiopsis showed rich genetic diversity. Inoculating the conidial suspension of one or both of the two species of the Pestalotiopsis on detached leaves or branches of M. rubra could cause lesions surrounding the inoculation sites with the frequency of 100 %. Moreover, necrotic lesions could be observed on inoculated potted plants with the frequencies of 33.3 % for P. mangiferae, 25 % for P. vismiae and 50 % for a mixed inoculum.     

A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100?fg^-1?pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well.     

Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland

Brown rot, caused by fungi belonging to the genus Monilinia, is one of the most important diseases of stone and pome trees in the world. During the summers of 2010 and 2011, a total of 670 Monilinia spp. isolates were obtained from infected fruits. They were collected from different commercial stone and pome fruit orchards, located in northern, southern and central Poland. All isolates were identified using multiplex PCR. Twenty isolates obtained from plum, peach and apple fruits were identified as M. polystroma and 5 isolates from plums as M. fructicola. The remaining isolates were identified as M. fructigena or M. laxa. The identification of the isolates was also confirmed on the basis of growth characteristics in culture according to the EPPO standard PM 7/18. A comparison of morphological features of four Monilinia spp. growing on two selective growth media, APDA-F500 and CHA, indicated significant differences between these species. In artificial inoculation of fruits, all the examined Monilinia spp. isolates were pathogenic. The species affiliation of M. polystroma and M. fructicola isolates collected from orchards in Poland was confirmed on the base of phylogenetic and sequence analysis of the internal transcribed spacer (ITS1/5.8S rDNA/ITS2) region of ribosomal DNA.     

蚕豆和豌豆锈病病原菌的分子鉴定

为明确我国热带和亚热带地区蚕豆Vicia faba和豌豆Pisum sativum锈病的病原菌种类,通过致病性测定和ITS序列系统发育分析对来自我国云南省玉溪市的4份豌豆锈菌分离物及云南、广西、重庆和四川省(区、市)的5份蚕豆锈菌分离物进行系统鉴定。结果显示,分离自豌豆的锈菌WX1分离物对蚕豆和豌豆均具有高致病性,在侵染叶片上产生大量锈子器;分离自蚕豆的锈菌CX3分离物仅对蚕豆具有高致病性,能在叶片上产生大量夏孢子,而对豌豆的致病性相对较低,仅产生少量的夏孢子堆;分离物WX1和CX3对小扁豆和鹰嘴豆不具有致病性。基于ITS序列系统发育分析表明,所有不同寄主来源的蚕豆单胞锈菌分离物均聚类于一个系统发育组,但分离自蚕豆和豌豆的分离物分别聚类在不同的亚组。表明分离自云南省玉溪市豌豆上的蚕豆单胞锈菌Uromyces viciae-fabae应为豌豆专化型,定名为U. viciae-fabae ex P. sativaum,而来源于云南、广西、重庆和四川省(区、市)的蚕豆锈病病原菌为蚕豆专化型U. viciae-fabae ex V. faba。     

Characterization of Phoma adonidicola causing a spot blight on Adonis palaestina

Adonis palaestina Boiss. is one of the top three natural sources of red pigment astaxanthin, which has been used as a valuable antioxidant nutraceutical and a feed additive for salmonid fish raising. Since 2004, a blight disease causing significant damage to plants of A. palaestina in Inner Mongolia, China has occurred. The disease caused small, brown lesions on petioles and stems of the host plants. The disease initially appeared in the field as a brown necrosis on lower parts of the plant, and eventually expanded to the whole plant resulting in complete defoliation. The fungus consistently isolated from symptomatic tissues was identified as Phoma adonidicola based on morphological characteristics. Phylogenetic analysis based on LSU (Large Subunit - 28S), ITS (ITS1-5.8S-ITS2 region), and TUB (β-tubulin) showed that the P. adonidicola isolates fall in the same well-supported clade, which is closely related to Stagonosporopsis ajacis. All isolates of P. adonidicola also caused typical spots on inoculated plants, and were successfully reisolated from the symptomatic tissues. The disease of A. palaestina was proposed as spot blight.     

Morphological and molecular characterization of Fusarium spp. associated with yellowing disease of black pepper (Piper nigrum L.) in Malaysia

Yellowing disease is one of the most important diseases of black pepper (Piper nigrum L.). To characterize the pathogen(s) responsible for yellowing disease of black pepper in Malaysia, 53 isolates of Fusarium were collected from the roots of diseased black pepper plants and from rhizosphere soils from major growing areas in Sarawak and Johor. A total of 34 isolates of F. solani and 19 isolates of F. proliferatum were obtained and identified based on morphological characteristics and molecular techniques. DNA sequencing of the internal transcribed spacers (ITS1 and ITS2) and 5.8S ribosomal DNA regions was conducted to identify Fusarium species. Nucleotide sequence analysis of the ITS regions revealed that this molecular technique enabled identification of Fusarium at the species level as F. solani and F. proliferatum. In a pathogenicity test on 3-month-old black pepper plants, F. solani was pathogenic, but F. proliferatum was not. On the basis of morphology, DNA sequences and pathogenicity of the fungal isolates from the diseased plants, we showed that yellowing disease on black pepper is caused by F. solani     

Occurrence of soilborne Phytophthora species in declining broadleaf forests in Hungary

Invasive Phytophthora species are responsible for severe tree diseases in many forest ecosystems in Europe. In Hungary, the symptoms were first noted when P. alni infection led to a serious decline and mortality of alder stands in the late 1990s. Between 2001 and 2009, over 300 soilborne Phytophthora isolates were collected from declining broadleaf forests in Hungary, and 10 Phytophthora species identified based on morphological traits and the molecular characteristics of the internally transcribed spacer (ITS) regions of the nuclear ribosomal DNA. The most diverse species spectrum, found in diseased alder stands, included P. gonapodyides, P. gregata, P. inundata, P. lacustris, P. megasperma, P. plurivora, one informally designated taxon: P. taxon hungarica, and one unnamed species P. sp.1. P. cactorum and P. plurivora isolates were prevalent in the soil of a declining eastern black walnut forest, and three species, P. gonapodyides, P. multivora and P. plurivora were recovered from a declining oak stand. More than one ITS-based genotype was identified for four species, including six genotypes for P. gonapodyides, and two each for P. cactorum, P. plurivora and P. inundata. The high genetic diversity of the P. gonapodyides isolates may indicate that the species is indigenous to the region. In contrast, the frequently recovered, widely distributed P. lacustris with a single ITS genotype may represent a recent colonizer. The P. multivora isolates are, to date, the first reported from a European native forest.     

新疆加工番茄腐霉根腐病病原鉴定及其rDNA的ITS区段分析

为了明确新疆加工番茄根腐病的病原种类,从成苗期到果实成熟期对各主栽区的病原物进行分离,并进行形态学鉴定和ITS序列分析.结果表明,从162个根腐病样中获得120个分离物,其中腐霉菌93株,占总分离物的77.5%.腐霉菌分离物鉴定为3个种,瓜果腐霉Pythium aphani-dermatum(Edson)Fitzp,56株,占腐霉分离物的60.22%;简囊腐霉P.monospermum Pringsheim,6株,占6.45%;棘腐霉P.acanthicum Drechsler,2株,占2.15%;另有29个分离物因未诱发出雄器和藏卵器而无法鉴定到种.     

我国部分地区玉米丝核菌组成及其致病类型分析

IA为主要融合群;双核丝核菌为AG-A融合群;单核丝核菌种类尚不确定.对各融合群的致病类型进行初步比较发现,属于AG1-IA融合群的菌株,可在玉米叶鞘形成典型的云纹状病斑,其它菌株虽可引起玉米发病,但与AG1-IA的症状存在明显差异.     

A strawberry disease caused by Acremonium strictum

We report Acremonium strictum as the causal agent of a new disease in strawberry plants (Fragaria x ananassa Duch.) in the Northwest of Argentina. Both the structure of conidiophores and the sequence spanning the internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA) allowed confirming the affiliation of the isolate, corresponding to A. strictum. An analysis of symptoms and lesions caused by the strain of A. strictum in susceptible cultivars showed that the typical symptoms are as follows: in an early stage, small necrotic light-brown spots in leaves and petioles increase in number and size as the disease progresses; in a more advanced stage, dark necrotic areas expand over petioles and leaves causing strangulation of petioles and the plant wilt. Crown rot was not observed even at a very advanced stage of the disease.     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。