First Report of Myrothecium roridum Causing Leaf Spot on Anthurium andraeanum in China

A new severe disease on Anthurium andraeanum Lind. was observed in the summer of 2011 in Beijing, China. The fungus was isolated from symptomatic leaves, and its pathogenicity was confirmed. Based on the morphological characteristics and molecular analysis, the pathogen was identified as Myrothecium roridum Tode ex Fr. This is the first report of M. roridum causing leaf spot on A. andraeanum in China.     

Occurrence of Charcoal Rot Caused by Macrophomina phaseolina,an Emerging Disease of Adzuki Bean in China

Adzuki bean (Vigna angularis) is an important legume crop in China. Soil‐borne charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is an important and devastating disease of many crops including legumes. During late August and early September, 2014, symptoms similar to charcoal rot were observed on adzuki bean plants in Yulin City of Shanxi Province, and Fangshan County of Beijing, China. This study was conducted to determine the causal agent of the emerging disease on adzuki bean. Four fungal isolates were obtained and identified as M. phaseolina based on morphological and molecular characteristics, including species‐specific primer detection and sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA. The resulting sequences showed 99% identity with more than 60 M. phaseolina strains from diverse hosts. The virulence on adzuki bean was verified using pathogenicity tests, producing symptoms similar to those observed in the fields. To our knowledge, this is the first report of M. phaseolina causing charcoal rot on adzuki bean.     

Somatic Hybrids Between Potato and S. berthaultii Show Partial Resistance to Soil‐Borne Fungi and Potato Virus Y

Three tetraploid somatic hybrid lines produced by protoplast fusion between a dihaploid potato, Solanum tuberosum, cultivar BF15 and the wild potato species Solanum berthaultii were evaluated here for their response to different soil‐borne pathogens, that is Fusarium solani, Pythium aphanidermatum and Rhizoctonia solani as well as to infection by potato virus Y (PVY). Both hybrid and BF15 plants grown in vitro were inoculated with the tested pathogen strains, that is R. solani, P. aphanidermatum, or F. solani. The growth level and disease severity index of these plants were compared to the susceptible commercial cultivar Spunta. A better growth of inoculated hybrid plants and restricted disease symptoms were observed in comparison with the commercial plants. Under glasshouse conditions and after inoculation with R. solani and P. aphanidermatum, improved resistance of the hybrid plants to these pathogens was confirmed. Indeed, these plants showed no significant damage following inoculation and a better development in R. solani‐infected plants. The susceptibility of the hybrid tubers to R. solani, P. aphanidermatum, and to F. solani infection was also determined. A significant reduction of tissue colonisation was observed in all the hybrid lines compared to the cultivated cultivars. The STBc and STBd hybrids also showed improved resistance to the PVY ordinary strain (PVY^o) under glasshouse conditions.     

Characterization and Pathogenicity of Phomopsis theicola Anamorph of Diaporthe foeniculina Causing Stem and Shoot Cankers on Sweet Chestnut in Italy

In March 2014, an outbreak of shoot cankers was observed on grafted Castanea sativa plants in a glasshouse in central Italy. Morphological characteristics led to the identification of isolates of Phomopsis recovered from cankered stems and shoots. Based on the morphological characteristics of colony appearance, shape of conidia and conidiomata as well as sequences of internal transcribed spacer regions (ITS), actin (ACT) and translation elongation factor (TEF‐1α), the fungus was identified as Phomopsis theicola/Diaporthe foeniculina. Pathogenicity test showed that P. theicola isolates were pathogenic to C. sativa when artificially inoculated, reproducing the symptoms originally observed. Koch's postulates were fulfilled by re‐isolating the pathogen. This is the first report of P. theicola/D. foeniculina causing stem and shoot cankers and dieback on C. sativa in Italy or elsewhere.     

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia‐recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH‐psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH‐psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.     

Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny

Benthic Prorocentrum species can produce toxins that adversely affect animals and human health. They are known to co‐occur with other bloom‐forming, potentially toxic, benthic dinoflagellates of the genera Ostreopsis, Coolia, and Gambierdiscus. In this study, we report on the presence of P. elegans M.Faust and P. levis M.A.Faust, Kibler, Vandersea, P.A. Tester & Litaker from the southeastern Bay of Biscay. Sampling was carried out in the Summer‐Autumn 2010–2012 along the Atlantic coast of the Iberian Peninsula, but these two species were only found in the northeastern part of the Peninsula. Strains were isolated from macroalgae collected from rocky‐shore areas bordering accessible beaches. Morphological traits of isolated strains were analyzed by LM and SEM, whereas molecular analyses were performed using the LSU and internal transcribed spacer (ITS)1‐5.8S‐ITS2 regions of the rDNA. A bioassay with Artemia fransciscana and liquid chromatography–high‐resolution mass spectrometry analyses were used to check the toxicity of the species, whose results were negative. The strains mostly corresponded to their species original morphological characterization, which is supported by the phylogenetic analyses in the case of P. levis, whereas for P. elegans, this is the first known molecular characterization. This is also the second known report of P. elegans.     

DNA barcoding of invasive plants in China: A resource for identifying invasive plants

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH‐psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra‐ and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH‐psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species.     

Evaluation of 11 single‐locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae)

The aim of this work was to evaluate the suitability of selected DNA regions in the barcoding of plants, based on the species belonging to the genus Lamium (Lamiaceae). For this purpose, nine chloroplast barcodes, that is, accD, matK, rbcL, rpoA, rpoB, rpoC1, rpoC2, trnH‐psbA, trnL‐trnF, as well as ITS nuclear region, and intron of mitochondrial nad5 gene were tested. Among the single‐locus barcodes, most effective in the identification of Lamium species was the trnH‐psbA spacer and matK gene. The high level of variability and resolving power was also observed in the case of rpoA and rpoC2 genes. Despite the high interspecies variability of ITS region, it turned out to be inapplicable in Lamium identification. An important disadvantage of ITS as a barcode is a limitation of its use in polyploid plants, samples contaminated with fungal material or samples with partially degraded DNA. We have also evaluated five‐two‐locus and two‐three‐locus barcode regions created from a combination of most effective single loci. The best‐performing barcode combinations were matK + trnH‐psbA and matK + rpoA. Both of them had equally high discriminative power to identify Lamium species.     

Internal Transcribed Spacer Sequence Analysis of Puccinia helianthi Schw. and Its Application in Detection of Sunflower Rust

Two basidiomycete‐specific primers ITS1‐F and ITS4‐B were used in identification of the genus Puccinia. The primers showed good specificity for the genus with an 816‐bp product that was amplified exclusively. Twenty sequences of internal transcribed spacer (ITS) regions of Puccinia helianthi isolates from China remain unchanged. The whole ITS length (including ITS1 sequence 194 bp, 5.8S rRNA gene 156 bp, ITS2 sequence 206 bp) was 556 bp. By comparing the aligned ITS sequences of several Puccinia isolates from China, Spain and the United States, ITS homogeneity among these sunflower rust isolates was >99%. Genetic homology and phylogeny of P. helianthi with other Puccinia spp. was investigated. Nineteen sequences of rDNA ITS1 and ITS2 were determined and used as phylogenetic markers. Phylogenetic analysis of ITS regions showed that Puccinia spp. of sunflower was clustered in one clade with P. komarovii and P. violae, divergent from Puccinia spp. of Chrysanthemum, P. tenaceti of tansy (Tanacetum vulgare) and Puccina spp. of big sagebrush (Artemisia tridentate) indicating sunflower rust had distant phylogenetic relationships with other Compositae rusts. With the specified primers SR‐1 and SR‐2, either from purified urediniospores or symptomless (but infected) sunflower leaves could be examined specifically. Therefore, results of this study help in detection and polygenetic study of rust fungi occurring on sunflower.     

First Report of Root‐knot Nematodes (Meloidogyne arenaria) on Angelica dahurica in China

Angelica dahurica is an important Chinese herbal medicine plant, and its rhizome is of high medicinal value. In recent years, a severe decline in yield has been observed in Bozhou City (China's largest A. dahurica producing area), Anhui province, China. It showed symptoms of decline, stunting, yellowing and many galls in the roots, which was the characterization of infestation by root‐knot nematodes. A survey of root‐knot nematodes on its roots was conducted in this area from June to September, 2011. Based on our results, the nematode species on A. dahurica was identified as Meloidogyne arenaria by the morphological, biochemical and molecular methods. To our knowledge, this is the first report of M. arenaria on A. dahurica in China.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.     

DNA barcoding of Corydalis,the most taxonomically complicated genus of Papaveraceae

The genus Corydalis is recognized as one of the most taxonomically challenging plant taxa. It is mainly distributed in the Himalaya–Hengduan Mountains, a global biodiversity hotspot. To date, no effective solution for species discrimination and taxonomic assignment in Corydalis has been developed. In this study, five nuclear and chloroplast DNA regions, ITS, ITS2, matK, rbcL, and psbA‐trnH, were preliminarily assessed based on their ability to discriminate Corydalis to eliminate inefficient regions, and the three regions showing good performance (ITS, ITS2 and matK) were then evaluated in 131 samples representing 28 species of 11 sections of four subgenera in Corydalis using three analytical methods (NJ, ML, MP tree; K2P‐distance and BLAST). The results showed that the various approaches exhibit different species identification power and that BLAST shows the best performance among the tested approaches. A comparison of different barcodes indicated that among the single barcodes, ITS (65.2%) exhibited the highest identification success rate and that the combination of ITS + matK (69.6%) provided the highest species resolution among all single barcodes and their combinations. Three Pharmacopoeia‐recorded medicinal plants and their materia medica were identified successfully based on the ITS and ITS2 regions. In the phylogenetic analysis, the sections Thalictrifoliae, Sophorocapnos, Racemosae, Aulacostigma, and Corydalis formed well‐supported separate lineages. We thus hypothesize that the five sections should be classified as an independent subgenus and that the genus should be divided into three subgenera. In this study, DNA barcoding provided relatively high species discrimination power, indicating that it can be used for species discrimination in this taxonomically complicated genus and as a potential tool for the authentication of materia medica belonging to Corydalis.     

Development of a Real‐time Fluorescence Loop‐mediated Isothermal Amplification Assay for Detection of Burkholderia gladioli pv. alliicola

Burkholderia gladioli pv. alliicola is a causal agent of rot on a wide range of hosts including onion and tulip. It is one of quarantine phytopathogenic bacteria in China. To reduce the economic losses associated with this pathogen, simple and rapid detection methods are needed. In this study, an efficient loop‐mediated isothermal amplification (LAMP) assay with a real‐time fluorometer was developed. The analysis of 16S‐23S rRNA intergenic transcribed spacer (ITS) sequences showed considerable variability between different Burkholderia species and B. gradioli pathovars. A set of LAMP primers was designed based on the ITS region. The sensitivity and specificity of the developed assay were evaluated at the optimal temperature of 65°C. The primers were specific for B. gladioli pv. alliicola and did not react to strains of others species and other pathovars in the species B. gladioli. The sensitivity of the real‐time LAMP assay was 1 fg DNA which was 100 times higher than that of conventional PCR. The method was verified by testing natural samples and inoculated onion seeds, and it showed effectiveness. The real‐time LAMP assay established in this study is an effective method for detection of B. gladioli pv. alliicola.     

Characterization of Botryosphaeria dothidea and Lasiodiplodia pseudotheobromae from English Walnut in China

China is the largest walnut producer in the world, and walnut trees, especially English walnut, are widely distributed in the country. Species of Botryosphaeriaceae include important plant pathogens that can cause diseases on many tree crops including English walnut. Recently, disease symptoms caused by Botryosphaeriaceae were observed on English walnut branches or kernels from Beijing, Henan and Sichuan provinces in China. Based on morphological characteristics and phylogenetic analyses of the ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, Botryosphaeria dothidea and Lasiodiplodia pseudotheobromae were identified. Pathogenicity tests showed that both species are virulent to English walnut. To our knowledge, this is the first report of L. pseudotheobromae infecting English walnut in the world.     

First Report of Sclerotinia nivalis Causing White Mold Disease on Sedum sarmentosum in China

In 2010 and 2011, a disease exhibiting characteristics of white mold was found on Sedum sarmentosum, a crassulaceous weed under canopies of tea trees, in Zhushan County, Hubei Province, China. Based on the cultural and morphological characteristics, the pathogen was identified as Sclerotinia nivalis Saito. In the phylogenetic tree inferred from the internal transcribed spacer (ITS)‐rDNA sequences, the pathogen was clustered with five previously characterized isolates of S. nivalis, forming a unique clade, thus confirming the morpho‐cultural identification. Koch’s postulates were fulfilled by pathogenicity tests using the isolate SsSn‐24 and Let‐19 of S. nivalis on plants of S. sarmentosum. To our knowledge, this is the first report of S. nivalis on S. sarmentosum in the family Crassulaceae.     

Trichobaris weevils distinguish amongst toxic host plants by sensing volatiles that do not affect larval performance

Herbivorous insects use plant metabolites to inform their host plant selection for oviposition. These host‐selection behaviours are often consistent with the preference–performance hypothesis; females oviposit on hosts that maximize the performance of their offspring. However, the metabolites used for these oviposition choices and those responsible for differences in offspring performance remain unknown for ecologically relevant interactions. Here, we examined the host‐selection behaviours of two sympatric weevils, the Datura (Trichobaris compacta) and tobacco (T. mucorea) weevils in field and glasshouse experiments with transgenic host plants specifically altered in different components of their secondary metabolism. Adult females of both species strongly preferred to feed on D. wrightii rather than on N. attenuata leaves, but T. mucorea preferred to oviposit on N. attenuata, while T. compacta oviposited only on D. wrightii. These oviposition behaviours increased offspring performance: T. compacta larvae only survived in D. wrightii stems and T. mucorea larvae survived better in N. attenuata than in D. wrightii stems. Choice assays with nicotine‐free, JA‐impaired, and sesquiterpene‐over‐produced isogenic N. attenuata plants revealed that although half of the T. compacta larvae survived in nicotine‐free N. attenuata lines, nicotine did not influence the oviposition behaviours of both the nicotine‐adapted and nicotine‐sensitive species. JA‐induced sesquiterpene volatiles are key compounds influencing T. mucorea females’ oviposition choices, but these sesquiterpenes had no effect on larval performance. We conclude that adult females are able to choose the best host plant for their offspring and use chemicals different from those that influence larval performance to inform their oviposition decisions.     

Resistance to Asparagus virus 1 in the Wild Relative Asparagus amarus

Five asparagus cultivars, three breeding lines and the wild relative Asparagus amarus were tested for natural infection by Asparagus virus 1 (AV‐1) in experimental fields at two locations over 3 and 4 years, respectively. In the first year after re‐planting the annual crowns in the field, more than 90% of tested plants of cultivars were infected by AV‐1. In the third and fourth year, 100% of tested plants of cultivars were AV‐1 infected. In comparison, all plants of the wild relative A. amarus were completely free of AV‐1, suggesting a high level of resistance. Additionally, 1‐year‐old glasshouse‐cultivated plants of A. officinalis and A. amarus were placed in an AV‐1 provocation cabin under field conditions. Seven months later, 100% of the A. officinalis plants showed a high virus concentration in ELISA, whereas no AV‐1 was detectable in the A. amarus plants. This result was confirmed by highly sensitive AV‐1‐specific RT‐PCR. To exclude vector resistance, the feeding behaviour of green peach aphid Myzus persicae was tested over 12 h using the electrical penetration graph method. Both asparagus genotypes were accepted by the aphids as potential hosts, but the feeding time was significantly longer on A. amarus. A genetic distance analysis of the various cultivars of Asparagus officinalis and selected wild relatives of the JKI collection was carried out, resulting in a clear discrimination of cultivars and wild relatives, especially A. amarus. The potential breeding value of the putative resistance carrier is discussed.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.     

Molecular data and ecological niche modelling reveal the Pleistocene history of a semi‐aquatic bug (Microvelia douglasi douglasi) in East Asia

This study investigated the Pleistocene history of a semi‐aquatic bug, Microvelia douglasi douglasi Scott, 1874 (Hemiptera: Veliidae) in East Asia. We used M. douglasi douglasi as a model species to explore the effects of historical climatic fluctuations on montane semi‐aquatic invertebrate species. Two hypotheses were developed using ecological niche models (ENMs). First, we hypothesized that M. douglasi douglasi persisted in suitable habitats in southern Guizhou, southern Yunnan, Hainan, Taiwan and southeast China during the LIG. After that, the populations expanded (Hypothesis 1). As the spatial prediction in the LGM was significantly larger than in the LIG, we then hypothesized that the population expanded during the LIG to LGM transition (Hypothesis 2). We tested these hypotheses using mitochondrial data (COI+COII) and nuclear data (ITS1 + 5.8S+ITS2). Young lineages, relatively deep splits, lineage differentiation among mountain ranges in central, south and southwest China and high genetic diversities were observed in these suitable habitats. Evidence of mismatch distributions and neutrality tests indicate that a population expansion occurred in the late Pleistocene. The Bayesian skyline plot (BSP) revealed an unusual population expansion that likely happened during the cooling transition between LIG and LGM. The results of genetic data were mostly consistent with the spatial predictions from ENM, a finding that can profoundly improve phylogeographic research. The ecological requirements of M. douglasi douglasi, together with the geographical heterogeneity and climatic fluctuations of Pleistocene in East Asia, could have shaped this unusual demographic history. Our study contributes to our knowledge of semi‐aquatic bug/invertebrate responses to Pleistocene climatic fluctuations in East Asia.