Taxonomy, phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world

Species of Botryosphaeria are well-recognized pathogens of pome and stone fruit trees. The taxonomy of these fungi, however, has been confused in the past. Recent taxonomic changes to the Botryosphaeriaceae further influence the literature pertaining to these fungi. This study reviews the taxonomic status of Botryosphaeriaceae associated with fruit tree diseases, identifies them in South Africa and elsewhere, and develops a reliable identification technique for them. Comparisons were made using DNA sequence data from the nuclear ITS rRNA operon and anamorph morphology. These analyses distinguished six clades amongst isolates associated with fruit tree diseases, corresponding to Neofusicoccum ribis (=  B. ribis ), N. parvum (=  B. parva ), N. australe (=  B. australis ), B. dothidea , Diplodia mutila (=  B. stevensii ) and ' Botryosphaeria ' obtusa (the genus Botryosphaeria is no longer available for the fungus known as B. obtusa , but a new name has not been proposed yet). Isolates from fruit trees in South Africa were grouped in the N. australe and ' Botryosphaeria' obtusa clades. This is the first report of N. australe from fruit trees. PCR-RFLP analysis using the restriction endonucleases Cfo I and Hae III distinguished the major clades. However, two groups of closely related species, N. ribis and N. parvum , and N. australe and N. luteum (=  B. lutea ), had identical RFLP profiles. Using RFLP, it was shown that ' Botryosphaeria ' obtusa is the dominant species on fruit trees in the Western Cape Province of South Africa. These results and methods will be useful in future epidemiological studies and disease management of Botryosphaeriaceae from fruit trees.     

芒果蒂腐病病原葡萄座腔菌科真菌种类鉴定

In order to identify the pathogen of Botryosphaeriaceae causing mango stem-end rot, tissue isolation method was used to process mango samples collected from Hainan, Guangxi, Yunnan, and Sichuan Provinces in China, and 58 fungal isolates were obtained. Among them, 39 isolates were belonged to Botryosphaeriaceae, accounting for 67.24%. These isolates were identified based on morphological characteristics and phylogenetic analysis with internal transcribed spacer (ITS) and elongation factor 1 alpha (EF-1α). The results showed that there were four main species of Botryosphaeriaceae, including Neofusicoccum parvum (33.33%), Lasiodiplodia theobromae (30.77%), Botryosphaeria dothidea (28.21%), and L. pseudotheobromae (7.69%). The mango stem-end rot caused by L. pseudotheobroma was firstly reported in China. The virulence of the four species was tested by in vitro inoculation and the results showed that all isolates were pathogenic, among which L. theobromae, L. pseudotheobroma, and N. parvum were the “strong” grade, and B. dothidea was the “middle” grade.     

Pathogenic Botryosphaeriaceae associated with <Emphasis Type="Italic">Mangifera indica</Emphasis> in the Kimberley Region of Western Australia

Members of the Botryosphaeriaceae, in particular Lasiodiplodia theobromae, Neofusicoccum parvum, N. mangiferum and Botryosphaeria dothidea, commonly cause stem cankers, dieback and stem end rot of mangoes worldwide. In the current study, eight taxa of Botryosphaeriaceae were identified as canker-associated fungi, pathogens, potential pathogens or endophytes of mangoes in the Kimberley, Australia. These include Neoscytalidium novaehollandiae, Ne. dimidiatum, Pseudofusicoccum adansoniae, P. ardesiacum, P. kimberleyense, Lasiodiplodia sp. 1, L. iraniensis and L. pseudotheobromae. The pathogenicity of a selection of these species toward fruit and branches was tested. All were pathogenic to mango in comparison to the control, with Lasiodiplodia spp. being the most pathogenic. It appears that either geographic isolation or the unique growing conditions in the Kimberley may have provided an effective barrier to the acquisition or establishment of known botryosphaeriaceous pathogens. Wounds caused by mechanical pruning may provide an entry point for infection, whilst severe pruning may increase plant stress.     

Characterization of Botryosphaeriaceae from plantation‐grown Eucalyptus species in South China

The Botryosphaeriaceae is a species‐rich family that includes pathogens of a wide variety of trees, including Eucalyptus species. Symptoms typical of infection by the Botryosphaeriaceae have recently been observed in Eucalyptus plantations in South China. The aim of this study was to identify the Botryosphaeriaceae associated with these symptoms. Isolates were collected from branch cankers and senescent twigs of different Eucalyptus spp. All isolates resembling Botryosphaeriaceae were separated into groups based on conidial morphology. Initial identifications were made using PCR‐RFLP fingerprinting, by digesting the ITS region of the rDNA operon with the restriction enzymes CfoI and KspI. Furthermore, to distinguish isolates in the Neofusicoccum parvum/N. ribis complex, a locus (BotF15) previously shown to define these species, was amplified and restricted with CfoI. Selected isolates were then identified using comparisons of DNA sequence data for the ITS rDNA and translation elongation factor 1‐alpha (TEF‐1α) gene regions. Based on anamorph morphology and DNA sequence comparisons, five species were identified: Lasiodiplodia pseudotheobromae, L. theobromae, Neofusicoccum parvum, N. ribis sensu lato and one undescribed taxon, for which the name Fusicoccum fabicercianum sp. nov. is provided. Isolates of all species gave rise to lesions on the stems of an E. grandis clone in a glasshouse inoculation trial and on the stems of five Eucalyptus genotypes inoculated in the field, where L. pseudotheobromae and L. theobromae were most pathogenic. The five Eucalyptus genotypes differed in their susceptibility to the Botryosphaeriaceae species suggesting that breeding and selection offers opportunity for disease avoidance in the future.     

珍珠李枝枯病病原鉴定及致病性测定

In order to confirm the pathogen of branch blight on pearl plum and its virulence, 14 samples were collected from Guangxi and Guizhou Province, and 21 isolates were obtained and were identified as Botryosphaeriaceae. The results of colony morphology, conidial observation and molecular identification of internal transcribed spacer region (ITS1-5.8S-ITS2) and the translation elongation factor 1-α (EFl-α) showed that the pathogens of branch blight on pearl plum were Botryosphaeria dothidea (66.67%), Neofusicoccum parvum (19.05%), N. algeriense (9.52%), and Lasiodiplodia pseudotheobromae (4.76%). The pathogenicity of different species of Botryosphaeriaceae was tested by mycelial dishes inoculation, and it was found that typical isolates of the four species were pathogenic, among which L. pseudotheobromae was the most virulent one and B. dothidea was the weakest one. This is the first report that L. pseudotheobromae causes branch blight on pearl plum.     

Incidence and distribution of botryosphaeriaceous species in New Zealand vineyards

The aim of this study was to determine the identification, incidence and distribution of botryosphaeriaceous species in New Zealand vineyards. A field study of 43 vineyards across six wine growing regions was conducted. A total of 336 isolates of botryosphaeriaceous species were isolated from 238 diseased grapevine samples. Morphological identification and phylogenetic analysis of the ribosomal RNA gene region, partial sequence of the translation elongation factor 1-alpha gene (EF1-α) and the β-tubulin gene identified nine botryosphaeriaceous species: N. parvum, N. luteum, N. australe, N. ribis, D. mutila, D. seriata, B. dothidea, Do. iberica and Do. sarmentorum. These species have been reported in other grape growing regions worldwide. Eighty eight percent of vineyards and 68% of symptomatic vines sampled were positive for botryosphaeriaceous species. Incidence and distribution of the botryosphaeriaceous species populations varied between the North and South Islands with N. parvum being the predominant species. The variability in incidence and distribution of the botryosphaeriaceous species may be influenced by climatic conditions and different sources of inoculum in the regions sampled. The results of this research clearly identified botryosphaeriaceous species as the target pathogens for development of management strategies for grapevine decline in New Zealand.     

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.     

Neofusicoccum eucalyptorum, a Eucalyptus pathogen, on native Myrtaceae in Uruguay

Neofusicoccum eucalyptorum is a canker-associated fungus apparently highly specialized on Eucalyptus . However, in surveys of the microbial population inhabiting native Myrtaceae in Uruguay, fungal cultures resembling N. eucalyptorum were isolated. The possible occurrence of N. eucalyptorum on hosts other than Eucalyptus prompted further investigation. Several surveys were conducted throughout Uruguay to obtain samples from native forests, focusing primarily on species in the Myrtaceae. Fungal identification was based on morphology and confirmed using comparison sequences for the internal transcribed spacer (ITS) of the rDNA operon. Pathogenicity was evaluated by inoculating plants of a Eucalyptus grandis clone. Morphological and molecular identification confirmed the occurrence of N. eucalyptorum on Blepharocalyx salicifolius, Myrceugenia glaucescens and Myrrhinium atropurpureum var. octandrum . This is the first report of N. eucalyptorum occurring in hosts other than Eucalyptus . Pathogenicity tests confirmed the ability of this species to produce cankers on E. grandis . This study provides information that will assist breeding programmes in attempts to obtain disease-resistant Eucalyptus plantations and it also suggests that a Eucalyptus pathogen could have moved to native trees in Uruguay.     

Detection of Botryosphaeriaceae species within grapevine woody tissues by nested PCR, with particular emphasis on the Neofusicoccum parvum/N. ribis complex

Several species of Botryosphaeriaceae and Phaeomoniella chlamydospora are common agents of grapevine decline worldwide. Currently, the use of culture independent PCR based techniques for detection of Botryosphaeriaceae within grapevine tissues has been limited to Botryosphaeria dothidea. In the present study, two Botryosphaeriaceae specific nested PCR assays were developed. One with a narrow target range, to detect Neofusicoccum parvum and the closely related species complex (Neofusicoccum parvum/N. ribis sensu Pavlic et al. Molecular Phylogenetics and Evolution 51:259–268, 2009) and another, with a wider range, to detect all 17 species of Botryosphaeriaceae which have been reported as potential wood pathogens of grapevine. The effectiveness of these assays was validated in vivo on naturally infected wood samples collected from standing vines and dormant grafted rooted cuttings commercialized in Italy by different nurseries in different years. All samples were also screened by means of a previously published nested PCR assay specific for Phaeomoniella chlamydospora. It was found that: 1) propagation material may play an important role as source of primary inoculum, not only of Phaeomoniella chlamydospora, as previously reported, but also for members of the Botryosphaeriaceae, among which Neofusicoccum parvum, Botryosphaeria dothidea and Diplodia seriata are the most common, and 2) multiple infections by different species belonging to Botryosphaeriaceae and/or Phaeomoniella chlamydospora occur frequently both in standing vines and propagation material. This last finding supports the hypothesis that at least some of the non-specific symptoms of grapevine decline may be due to the presence of different pathogens within host tissues.     

桉树溃疡病病原菌的鉴定

 随着桉树人工纯林的面积扩大,桉树病害也日趋加重,严重影响和制约桉树产业的发展。2008年至今,在云南省石林县和红河州桉树种植区发生一种病害,主要为害嫩枝、枝条和主干,发病初期,感病枝条和主干上产生红褐色圆或椭圆形坏死斑,以后逐渐扩大成椭圆或不规则溃疡斑,病变扩展到木质部后,常造成枝条生长畸形,易引起刮风断裂。主干树皮下明显呈黑褐色坏死,并造成感染面周围树皮开裂,溃疡处一般可看到流胶,严重时造成整株干枯死亡。调查发现,该病发病率通常在 10％～25％,重者可达 40％ 以上,引起桉树溃疡和枯萎死亡。本文通过病原菌分离、致病性测定及传统、分子鉴定明确了该病的病原,以期为病害防治提供理论依据。     

蓝莓枝枯病病原菌鉴定

 蓝莓 (Vaccinium spp.),是一类属于杜鹃花科 (Ericaceae) 越橘属 (Vaccinium) 的多年生小浆果类果树,广泛地应用于医药、保健、化妆品和环境保护等各方面,联合国粮农组织将其果实列为人类世界五大健康食品之一,也是我国南方酸性土丘陵地区值得发展的经济作物。全国已超过 10 个省份开始大面积的蓝莓商业化栽培,预计未来 10 年将超过 10 万 hm^{2 [1]}。但是,随着生产面积的不断扩大,蓝莓病害也日趋严重,严重影响和制约蓝莓产业的发展。2010年至今,在云南省昆明市周边及丽江市蓝莓种植区发生一种病害,主要为害嫩枝、枝条和主干,发病初期,感病枝条上产生红褐色圆或椭圆形坏死斑,随后逐渐扩大,造成枝条干枯死亡,并可扩展造成主干枯萎。调查发现,该病发病率通常在 10％～20％,重者可达 25％ 以上,可引起蓝莓整株枯萎死亡。本文通过病原菌分离、致病性测定及传统、分子鉴定,明确了该病的病原,以期为病害防治提供理论依据。     

Discovery of the Eucalyptus canker pathogen Chrysoporthe cubensis on native Miconia (Melastomataceae) in Colombia

Chrysoporthe cubensis is one of the most serious canker pathogens on commercially grown Eucalyptus species in the tropics and subtropics. During recent surveys for native hosts of C. cubensis in Colombia, fungi with fruiting structures similar to those of C. cubensis were found on native Miconia theaezans and Miconia rubiginosa , both members of the Melastomataceae. These fungi were identified based on morphology and DNA sequences of the ITS1/ITS2 region of the rDNA operon and the β-tubulin genes. The majority of isolates from M. theaezans and M. rubiginosa grouped together with South American C. cubensis isolates from Eucalyptus species and Syzygium aromaticum (clove). However, some of the isolates from M. theaezans grouped with isolates of Chrysoporthella hodgesiana , another anamorph species linked to Chrysoporthe , from Tibouchina spp. in Colombia. Pathogenicity of these fungi was assessed on various Melastomataceae. Miconia rubiginosa was more susceptible to infection by C. cubensis than two Eucalyptus clones . Isolates of C. cubensis and Chrysop. hodgesiana were mildly pathogenic on the various hosts included in the pathogenicity trials, and most pathogenic on Tibouchina urvilleana and Tibouchina lepidota .     

Canker and die-back of Eucalyptus in South Africa caused by Botryosphaeria dothidea

Die-back and canker of several Eucalyptus species and clones were observed during a survey of the most important forestry areas of South Africa. Disease was often associated with extreme environmental conditions such as drought, hot winds and frost. Botryosphaeria dothidea , which is well known as a cause of canker and die-back of Eucalyptus in other countries, was consistently isolated from symptomatic trees. Artificial inoculations of Eucalyptus nitens resulted in lesion development, confirming the pathogenicity of 5. dothidea to Eucalyptus in South Africa. This is the first report of this disease in South Africa.     

A Preliminary Investigation of Pathogenic Fungi from Lotus (Nelumbo nucifera Gaertn)in Nanchang City

[Objectives] Lotus (Nelumbo nucifera Gaertn) is an economically important aquatic plant in China. Fungaldisease is a serious problem in lotus cultivation. In this study, the pathogenic fungi on lotus in Nanchang City were investigated to lay the foundation for the disease control. [Methods] Lotus leaves and stems in ponds of Nanchang City were collected, the fungi on leave/stem spots were isolated and purified. Colonies morphological characters and ITS sequences were used to identify the strains. [Results] 49 strains were isolated and identified to 20 species, belonging to 12 genera. [Discussion] 15 species may firstly be reported on lotus in this study, i.e., Alternaria angustiovoidea, Alternaria compacta, Alternaria ricini, Alternaria tenuissima, Arthrinium arundinis, Botryosphaeria dothidea, Curvularia spicifera, Diaporthe australiana, Diaporthe eres, Diaporthe tectonae, Epicoccum nigrum, Fusarium fujikuroi, Neofusicoccum parvum, Nigrospora sphaerica, and Phomopsis eucommii.     

Botryosphaeriaceae species causing dieback on Annonaceae in Brazil

In Brazil, the Annonaceae species Annona muricata, A. squamosa, A. cherimola and atemoya (a hybrid of A. cherimola and A. squamosa) are cultivated in several regions, and produce fruits that are highly appreciated by consumers and are of great economic importance. Among the several diseases that can affect these crops, dieback is one of the most important, causing damage and, in the most severe cases, death of the plants. Due to the lack of suitable diagnostic studies up to now, this work aimed to identify the Botryosphaeriaceae species that cause dieback on Annonaceae in Brazil. Based on combined phylogenetic analyses of ITS, TEF-1α, TUB2 and RPB2, eight species of Botryosphaeriaceae were identified, namely Lasiodiplodia brasiliense, L. crassispora, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. subglobosa, L. theobromae and Pseudofusicoccum stromaticum. All species found in this study were pathogenic and caused symptoms of necrosis in stems and dieback. Thus, this study confirms species of Botryosphaeriaceae as causal agents of dieback on Annonaceae in Brazil.     

Ceratocystis species infecting stem wounds on Eucalyptus grandis in South Africa

Reports of Ceratocystis spp. causing disease of exotic plantation hardwood species have increased in recent years. Ceratocystis fimbriata causes wilt and canker on Eucalyptus spp. in Africa and South America, and C. albofundus results in wilt and death of Acacia mearnsii in Africa. Ceratocystis spp. generally infect wounds on trees, and artificial stem wounding can thus be used to determine the presence of these fungi. The aim of this study was to identify Ceratocystis spp. infecting wounds on Eucalyptus grandis in South Africa. Isolated Ceratocystis spp. were identified using morphological characteristics and comparisons of DNA sequence data for the ITS and 5·8S regions of the rRNA operon. Pathogenicity trials were conducted in the greenhouse to determine the possible role that these Ceratocystis spp. could have in disease development. These trials were also conducted under field conditions. Three Ceratocystis spp. were collected: C. fimbriata , C. moniliformis and C. pirilliformis . This is the first report of C. fimbriata and C. pirilliformis from Eucalyptus spp. in South Africa, and the first report of the latter fungus outside Australia. Both C. fimbriata and C. pirilliformis caused significant lesions on inoculated E. grandis trees. This is the first evidence that C. pirilliformis is a pathogen of Eucalyptus spp. From the results of both greenhouse and field trials, it has the potential to cause serious disease problems in Eucalyptus plantations.     

Inhibitory activity against plant pathogenic fungi of extracts from Myoporum bontioides A. Gray and indentification of active ingredients

BACKGROUND: In order to understand the bioactivity of Myoporum bontioides A. Gray against plant pathogens and determine its active ingredients, the inhibitory activities of methanol extracts from M. bontioides against Fusarium oxysporum f. sp. niveum (E. F. Smith) Snyder & Hansen, Pestalotia mangiferae P. Henn., Thielaviopsis paradoxa (De Seynes) v. Hohnel, Colletotrichum musae (Berk. & M. A. Curtis) v. Arx, Alternaria alternata (Fr.) Keissler, Mycosphaerella sentina (Fr.) Schroter and Sphaceloma fawcettii Jenk. were evaluated using a growth rate method, and the active ingredient was isolated by activity-directed isolation and identified by determination and analysis of IR, (1)H NMR, (13)C NMR and mass spectra and correlative physical constants. RESULTS: The results showed that the extracts from stems and leaves of M. bontioides exhibited inhibitory activity against the seven fungi, with > 58% inhibition at 10 g L(-1) after 72 h. The active compound was isolated and identified as (-)-epingaione, and showed inhibitory activity against the above seven fungi. The inhibitory activity against P. mangiferae was the highest, with an EC(50) value of 77 mg L(-1). The EC(50) values against the other six fungi were 147-245 mg L(-1). (-)-Epingaione also inhibited spore germination of F. oxysporum f. sp. niveum, T. paradoxa and S. fawcettii.CONCLUSION: (-)-Epingaione demonstrated broad-spectrum inhibitory activity against plant pathogenic fungi and is promising for exploitation as a fungicide.     

Botryosphaeriaceae species associated with blueberry dieback and sources of primary inoculum in propagation nurseries in New Zealand

Sampling at blueberry farms found Botryosphaeriaceae fungi in five of seven farms sampled; overall incidence was 41.4%, with Neofusicoccum australe (79.0%), N. luteum (8.0%), N. ribis (8.0%) and N. parvum (5.0%). Sampling of nursery plants found infections in all four nurseries with 45% incidence in mainly asymptomatic plants, which were infected with N. australe (66.0%), N. parvum (31.5%) and N. ribis (2.5%). Asymptomatic propagation cuttings from one nursery were found to have external contamination of Botryosphaeriaceae DNA (90.0%) and internal infection (65.0%) by the four main species found in the blueberry farms and nurseries. For propagation media nested PCR showed that out of the 98 samples received, 43 samples were positive for the presence of Botryosphaeriaceae DNA (44.0%). Results from the SSCP indicated that N. australe, N. luteum, N. parvum/ N. ribis and Diplodia mutila were present in the propagation media received. Isolates of the four main species recovered from farms and nurseries were pathogenic on blueberry stems but pathogenicity differed significantly between species and isolates within a species, with N. ribis being the most pathogenic, then N. parvum, N. luteum and N. australe. Overall, the high rate of infection in nursery plants indicated that nurseries can be a major source of infection for blueberry farms. Since propagating cuttings are the likely sources of infection for nurseries, these should be targeted in the control strategies.     

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.