Isolation and Identification of Pathogens Causing Marigold (Tagetes erecta L.) Black Spot in Beijing,China

Black spot leads to great marigold losses worldwide. The disease is characterized by black spots on leaves and stems in its early stages, and the whole plant has black rot at the advanced stage. In this report, 6 of 217 Alternaria strains isolated from lesions of marigold plants in Beijing were randomly selected. The morphological characteristics and a pathogenic tree based on two protein‐coding genes (gpd and alt a 1) indicated that Alternaria tagetica is the causal agent of marigold black spot in Beijing. All six Alternaria strains could successfully re‐infect marigold, but they could not infect carrot or zinnia by either spore spray in a greenhouse or planting experiments in the epidemic area. This is the first report of the A. tagetica pathogen being isolated from marigold in Beijing.     

First Report of Alternaria Leaf Spot on Gerbera (Gerbera jamesonii H. Bolux ex J. D. Hook) in Bulgaria

Alternaria leaf spots of gerbera (Gerbera jamesonii H. Bolus ex J. D. Hook) were observed on plants from different greenhouses on commercial plants in Bulgaria. The symptoms of the disease on the leaves were characterized by the development of brown, small, scattered dots, which gradually enlarged and coalesced to form large, oval, circular or irregular, brown to black lesions with concentric rings. Affected plants showed lower vitality, suppressed development and fewer, smaller, distorted in shape flowers. Alternaria isolates, obtained from infected leaf tissues were grown in pure culture and the morphological characteristics of the colony and sporulation apparatus were determined. DNA, extracted from the fungal isolates was subjected to polymerase chain reaction (PCR) with primers ITS1/ITS4, amplifying the internal transcribed spacer (ITS) regions. The resulting products were sequenced and compared for homology with other species in the GeneBank. The isolates showed 94% homology of the ITS region with either Alternaria alternata, A. arborescens, A. tenuissima, A. longipes, A. lini or A. smyrnii. None of the studied isolates was amplified with the A. alternata specific primers AAF2/AAR3, indicating that they are pathogenic varieties of it or belong to another species. Pathogenicity tests on 10 gerbera cultivars revealed that all of them were susceptible to Alternaria leaf spot. Additional tests on nine other crops (Solanum lycopersicum, Calendula officinalis, Capsicum annuum, Celosia argantea, Pelargonium spp., Petunia hybrida, Nicotiana tabacum, Cucurbita moscata and Raphanus sativus var. radicina) and on tomato or pepper fruits, potato tubers and carrot roots also indicated that all tested plant species were potential hosts of the disease. This is the first report of highly virulent isolates of Alternaria spp. in Bulgaria that cause leaf spots on gerbera in greenhouses.     

Alternaria on cruciferous plants. 4.Alternaria species on seed of some cruciferous crops and their pathogenicity

The incidence ofAlternaria spp. on seed samples of cruciferous vegetable crops was surveyed between 1990 and 1992. Some commercial seed lots of crucifers which are commonly grown in Japan were infested withAlternaria species. ThreeAlternaria species were encountered on the seed samples ofBrassica campestris, B. orelacea, andRaphanus sativus. The most frequently detected species wereA. japonica andA. alternata onB. campestris, A. brassicicola onB. oleracea, andA. japonica andA. alternata onR. sativus, respectively.Alternaria brassicae was not detected in this study.Alternaria brassicicola isolates from these crops produced necrotic lesions on all of the crucifer seedlings inoculated, whileA. japonica induced different reactions in different plants or plant parts depending on isolates used in inoculation tests. In contrast, most isolates ofA. alternata could not produce necrotic lesions on foliage leaves of crucifers inoculated, although some of them produced clear lesions only on cotyledons.Alternaria alternata associated with these cruciferous crop seeds was considered to be an oppotunistic parasite of these crops.     

Phylogeny of the Highly Divergent Echinosteliales (Amoebozoa)

Myxomycetes or plasmodial slime molds are widespread and very common soil amoebae with the ability to form macroscopic fruiting bodies. Even if their phylogenetic position as a monophyletic group in Amoebozoa is well established, their internal relationships are still not entirely resolved. At the base of the most intensively studied dark‐spored clade lies the order Echinosteliales, whose highly divergent small subunit ribosomal (18S) RNA genes represent a challenge for phylogenetic reconstructions. This is because they are characterized by unusually long variable helices of unknown secondary structure and a high inter‐ and infraspecific divergence. Current classification recognizes two families: the monogeneric Echinosteliaceae and the Clastodermataceae with the genera Barbeyella and Clastoderma. To better resolve the phylogeny of the Echinosteliales, we obtained three new small subunit ribosomal (18S) RNA gene sequences of Clastoderma and Echinostelium corynophorum. Our phylogenetic analyses suggested the polyphyly of the family Clastodermataceae, as Barbeyella was more closely related to Echinostelium arboreum than to Clastoderma, while Clastoderma debaryanum was the earliest branching clade in Echinosteliales. We also found that E. corynophorum was the closest relative of the enigmatic Semimorula liquescens, a stalkless‐modified Echinosteliales. We discuss possible evolutionary pathways in dark‐spored Myxomycetes and propose a taxonomic update.     

Quantification of Alternaria,Cladosporium, Fusarium and Penicillium verrucosum in Conventional and Organic Grains by qPCR

We analysed the levels of Alternaria, Cladosporium, Fusarium and Penicillium verrucosum in grain samples harvested in 2011 and 2012 from conventional and organic farms using qPCR. In general, both Alternaria and Cladosporium occurred in all cereal grains in the highest quantities, followed by P. verrucosum and Fusarium. Alternaria, Cladosporium and P. verrucosum had the highest levels in crop mixtures, barley and rye and lower levels in wheat, while Fusarium levels were the highest in crop mixtures and wheat. The levels of Alternaria and P. verrucosum were higher in organic rye and wheat than conventional grains. Although the level of Fusarium was higher in conventional than organic rye, opposite results were obtained for crop mixtures. A positive correlation was found between Alternaria, Cladosporium and P. verrucosum, indicating that similar factors might affect the distribution of these fungi in grains.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

First report of Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler on Sonchus oleraceus L. and Convolvulus arvensis L. in Algeria

Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler was found on sow thistle (Sonchus oleraceus L.) and field bindweed (Convolvulus arvensis L.) in the experimental station of ENSA (Ecole Nationale Supérieure d'Agronomie) in Algiers, Algeria, during the winter of 2016. Necrotic spots in the form of concentric circles were observed on the leaves of both weeds with disease incidence of approximately 70% and disease severity ranging from 50% to 70%. Fungi were isolated from the infected leaves and identified as A. alternata, based on morphological and molecular analyses (using genetic marker internal transcribed spacer, ITS of rDNA). Pathogenicity tests confirmed that A. alternata is the causing agent of leaf spot disease of sow thistle and field bindweed in accordance with the original symptoms. To the best of our knowledge, this is the first report of sow thistle and field bindweed naturally infected by A. alternata in Algeria.     

Morphology,phylogeny and pathogenicity of Alternaria species,involved in leaf spot disease of sunflower in northern Iran

Leaf spot disease of sunflower is one of the most important foliar diseases on this crop worldwide. Several fungal groups are known to cause leaf spot disease on sunflower. Species of the genus Alternaria are the most common and serious leaf spot causing fungi on this crop. Leaf spot disease is the most destructive foliar diseases on sunflower in northern Iran; however, the identity of the causal agent remains unknown. The present study was aimed to characterise the identity of the causal agent of the disease by means of morphological and molecular data as well as to evaluate the pathogenicity of the responsible species. For this purpose, a total number of 97 fungal isolates were recovered from sunflower leaves with leaf spot disease symptoms from the sunflower fields in northwestern zone of Iran. All of the isolates were identified as Alternaria alternata based on cultural and morphological characteristics. A subset of isolates was subjected to phylogenetic analysis using sequence data from ITS-rDNA region, gpd and rpb2 genes. Sequence data from ITS-rDNA and gpd did not discriminate A. alternata from the other small-spored Alternaria species. A phylogeny inferred using sequence data from rpb2 gene clustered our isolates in several sub-clades within a single monophyletic clade. Sequence data for the type strain of the other small-spored Alternaria species has to be included in phylogenetic analysis, in order to make sure, whether the observed variations in rpb2 gene sequences are an indication for the population variation in sunflower isolates of A. alternata or define species boundaries among the small-spored Alternaria species. The results of pathogenicity assay on sunflower plants (cultivar Euroflor) under greenhouse condition revealed that A. alternata is pathogenic on sunflower.     

Antiproliferative,Antifungal, and Antibacterial Activities of Endophytic Alternaria Species from Cupressaceae

Recent research has shown the bioprospecting of endophytic fungi from Cupressaceae. Here, we further uncover that the healthy cypress plants such as Cupressus arizonica, Cupressus sempervirens var. cereiformis, and Thuja orientalis host highly bioactive endophytic Alternaria fungal species. Indeed, endophytic Alternaria alternata, Alternaria pellucida, and Alternaria tangelonis were recovered from healthy Cupressaceous trees. Biodiversity and bioactivity of recovered endophytic Alternaria species were a matter of biogeography and host identity. We further extracted such Alternaria’s metabolites and highlighted their significant antiproliferative, growth inhibitory, and antibacterial activities against the model target fungus Pyricularia oryzae and the model pathogenic bacteria Bacillus sp., Erwinia amylovora, and Pseudomonas syringae. In vitro assays also indicated that endophytic Alternaria species significantly inhibited the growth of cypress fungal phytopathogens Diplodia seriata, Phaeobotryon cupressi, and Spencermartinsia viticola. In conclusion, since the recovered Alternaria species were originally reported as pathogenic and allergenic fungi, our findings suggest a possible ecological niche for them inside the foliar tissues of Cupressaceous trees. Moreover, in this study, the significant bioactivities of endophytic Alternaria species in association with Cupressaceae plant family are reported.     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Arthrinium phaeospermum,Phoma cladoniicola and Ulocladium consortiale,New Olive Pathogens in Italy

In recent years, leaf necrosis and twig dieback in the olive crop have been detected in Sicily (Italy). In this article, we identify the predominant fungal species associated with symptomatic leaves and twigs, using morphological features and DNA sequencing of the internal transcribed spacer (ITS) region, as Alternaria alternata, Arthrinium phaeospermum, Phoma cladoniicola and Ulocladium consortiale. The pathogenicity of these four species was tested on olive plants cv. Biancolilla. All species were pathogenic on leaves, but only U. consortiale produced cortical lesions on twigs, thus suggesting its main role in the Olea europaea twig dieback. To our knowledge, this is the first report of A. phaeospermum, P. cladoniicola and U. consortiale as olive pathogens.     

Pathogenic Variability within Indian Alternaria brassicae Isolates Using Seed,Cotyledon and Leaf of Brassica Species

Thirty Alternaria brassicae (Berk.) Sacc. isolates from diverse geographical locations of India were studied for pathogenic variability on seed, cotyledon and true leaves of Brassica species. Seed germination was reduced maximum by isolate BAB‐39 in Brassica juncea cultivar Varuna (22.1%), Brassica rapa var. Toria cultivar PT‐303 (12%), Brassica carinata cultivar Kiran (12%), Brassica napus cultivar GSL‐1 (11%) and tolerant source of B. juncea genotype PHR‐2 (7%), although least by isolate BAB‐49. Maximum lesion size on leaf was recorded in B. juncea cultivar Rohini (11.2, 16.5 and 16.8 mm) with isolates BAB‐09 (Pantnagar), BAB‐19 (Bharatpur) and BAB‐39 (Kangra), respectively, and categorized as highly virulent, while minimum lesion size of 3.2, 3.7 and 3.8 mm was observed with isolates BAB‐47 (Tonk), BAB 49 (Jobner) and BAB 04 (Kamroop), respectively, considered the weak isolates. On B. alba, BAB‐09, BAB‐19 and BAB‐39 isolates caused maximum lesion size of 3.7, 3.8 and 3.9 mm, respectively, even though it showed maximum tolerance. In both seed and cotyledon inoculation method, the per cent Alternaria blight severity above 80% was observed with isolate of BAB‐39 (91.5%), BAB‐19 (89.0%), BAB‐09 (85.5%) and least in isolate BAB‐49 (34.0%). Brassica seed, cotyledon and leaf showed the similar positive response for categorizing A. brassicae isolates as virulent and avirulent. This information could be used to the development and assessment of resistant brassica germplasm, especially with A. brassicae populations exhibiting increased virulence.     

Identification of Fungal Species Associated with Cladode Spot of Prickly Pear and Their Sensitivity to Chitosan

México is the most important producer of prickly pear (Opuntia ficus‐indica) in the world. There are several fungal diseases that can have a negative impact on their yields. In this study, there was a widespread fungal richness on cladodes spot of prickly pears from México. A total of 41 fungi isolates were obtained from cladodes spot; 11 of them were morphologically different. According to the pathogenicity test, seven isolates caused lesions on cladodes. The morphological and molecular identification evidenced the isolation of Colletotrichum gloeosporioides, Alternaria alternata, Fusarium lunatum, Curvularia lunata. All these species caused similar symptoms of circular cladodes spot. However, it is noticeable that some lesions showed perforation and detachment of affected tissues by Fusarium lunatum. To our knowledge, this is the first report of the Fusarium lunatum as phytopathogenic fungus of cladodes of prickly pear. The chitosan inhibited the mycelium growth in the seven isolates of phytopathogenic fungi. Chitosan applications diminished the disease incidence caused by C. gloeosporioies and F. lunatum in 40 and 100%, respectively. Likewise, the lesion severity index in cladodes decreased. There are no previous reports about the application of chitosan on cladodes of prickly pears for the control of phytopathogenic fungi. Therefore, this research could contribute to improve the strategies for the management of diseases in prickly pear.     

Cucumis sativus and Ipomoea purpurea: New hosts for Alternaria hydrangeae in China

Alternaria leaf spot is a common disease on various plants worldwide. In this study, an Alternaria species, A. hydrangeae causing leaf spot on Cucumis sativus (cucumber) and Ipomoea purpurea in China was identified based on morphology and multi-locus analysis of the partial ITS, GAPDH, Alt a 1, TEF1, and RPB2 gene regions. The pathogenicity of the present isolates and a representative isolate of A. hydrangeae were assessed on living leaves of C. sativus, I. purpurea and Hydrangea paniculata. Similar symptoms were observed on every plant inoculated with the isolates. The same fungus was re-isolated from inoculated leaves fulfilling Koch's postulates. Morphology of the original and re-isolated isolates showed characteristics similar to A. hydrangeae with some variations. Multi-locus analysis indicated that the present isolate fell into A. hydrangeae clade. This study is the first report of A. hydrangeae as an agent of Alternaria leaf spot in C. sativus and I. purpurea in China, which extends the host range of the fungus.     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.     

Invasive submerged freshwater macrophytes are more plastic in their response to light intensity than to the availability of free CO2 in air‐equilibrated water

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Multi‐locus phylogeny of African pipits and longclaws (Aves: Motacillidae) highlights taxonomic inconsistencies

The globally distributed avian family Motacillidae consists of five to seven genera (Anthus, Dendronanthus, Tmetothylacus, Macronyx and Motacilla, and depending on the taxonomy followed, Amaurocichla and Madanga) and 66–68 recognized species, of which 32 species in four genera occur in sub‐Saharan Africa. The taxonomy of the Motacillidae has been contentious, with variable numbers of genera, species and subspecies proposed and some studies suggesting greater taxonomic diversity than currently recognized (five genera and 67 species). Using one nuclear (Mb) and two mitochondrial (cyt b and CO1) gene regions amplified from DNA extracted from contemporary and museum specimens, we investigated the taxonomic status of 56 of the currently recognized motacillid species and present the most taxonomically complete and expanded phylogeny of this family to date. Our results suggest that the family comprises six clades broadly reflecting continental distributions: sub‐Saharan Africa (two clades), the New World (one clade), Palaearctic (one clade), a widespread large‐bodied Anthus clade, and a sixth widespread genus, Motacilla. Within the Afrotropical region, our phylogeny further supports recognition of Wood Pipit Anthus nyassae as a valid species, and the treatment of Long‐tailed Pipit Anthus longicaudatus and Kimberley Pipit Anthus pseudosimilis as junior subjective synonyms of Buffy Pipit Anthus vaalensis and African Pipit Anthus cinnamomeus, respectively. As the disjunct populations of Long‐billed Pipit Anthus similis in southern and East Africa are genetically distinct and geographically separated, we propose a specific status for the southern African population under the earliest available name, Nicholson's Pipit Anthus nicholsoni. Further, as our analyses indicate that Yellow‐breasted Pipit Anthus chloris and Golden Pipit Tmetothylacus tenellus are both nested within the Macronyx longclaws, we propose transferring these species to the latter genus.     

Similarities and spatial variations of bacterial and fungal communities in field rice planthopper (Hemiptera: Delphacidae) populations

Rice planthoppers are notorious plant sap‐feeding pests which cause serious damage. While several microbes in rice planthoppers have been broadly characterized, the abundance and diversity of bacteria and fungi in field planthoppers are largely unknown. This study investigated the bacterial and fungal community compositions of Chinese wild rice planthoppers Laodelphax striatellus and Sogatella furcifera using parallel 16S rRNA gene amplicon and internal transcribed space region sequencing. The bacteria varied significantly between the species and were partitioned significantly by sex, tissues and host environments in each species. The majority of bacteria were affiliated with the genera Wolbachia, Cardinium, Rickettsia and Pantoea. The abundance of Wolbachia was negatively correlated with that of Cardinium in both planthopper species. Compared with bacteria, the abundance and diversity of fungi did not differ between sexes but both were enriched in the gut. The bacterial community as a whole showed no significant correlation with the fungal community. The majority of fungi were related to Sarocladium, Alternaria, Malassezia, Aspergillus and Curvularia. A phylogenetic analysis revealed that these fungi were closely related to botanic symbionts or pathogens. Our results provide novel insights into the bacteria and fungi of rice planthoppers.