Characterization of Alternaria species associated with potato foliar diseases in China

The population structure of Alternaria species associated with potato foliar diseases in China has not been previously examined thoroughly. Between 2010 and 2013, a total of 511 Alternaria isolates were obtained from diseased potato leaves sampled in 16 provinces, autonomous regions or municipalities of China. Based on morphological traits and molecular characteristics, all the isolates were identified as Alternaria tenuissima, A. alternata or A. solani. Of the three species, A. tenuissima was the most prevalent (75·5%), followed by A. alternata (18·6%) and A. solani (5·9%). Phylogenetic analysis based on sequences of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) of representative Alternaria isolates showed that A. solani was distinct from the two small‐spored Alternaria species. Phylogenetic analysis of the partial coding sequence of the histone 3 gene divided the same collection of isolates into three main clades representing A. tenuissima, A. alternata and A. solani, respectively. The pathogenicity of the isolates on detached leaves of potato cv. Favorite did not differ significantly between the three species or between isolates from different geographical origins. The results indicate that the population structure of Alternaria species associated with potato foliar diseases differs from that reported previously in China. This is the first report of A. tenuissima causing potato foliar diseases in China.     

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.     

Incidence,pathogenicity and diversity of Alternaria spp. associated with alternaria leaf spot of canola (Brassica napus) in Australia

Studies were undertaken to determine Alternaria spp. associated with leaf spot symptoms on canola (Brassica napus) in two cropping seasons (2015, 2016) across southern Australia. Major allergen Alt a1 and plasma membrane ATPase genes were used to identify Alternaria spp. In 2015, 112 isolates of seven Alternaria spp. were obtained, with A. metachromatica predominating. In 2016, 251 isolates of 12 Alternaria spp. were obtained, with A. infectoria predominating. Alternaria spp. isolates were morphologically and phylogenetically identified and studies to determine their pathogenicity on both B. napus (cv. Thunder TT) and B. juncea (cv. Dune) confirmed 10 species (A. alternata, A. arborescens, A. brassicae, A. ethzedia, A. hordeicola, A. infectoria, A. japonica, A. malvae, A. metachromatica and A. tenuissima) as pathogenic on both Brassica species. Alternaria ethzedia, A. hordeicola and A. malvae were recorded for the first time in Australia on any host and the record of A. arborescens was the first for New South Wales (NSW) and South Australia (SA). Other first records included A. infectoria on B. napus in NSW; A. japonica on B. napus in NSW and Western Australia (WA); A. metachromatica on any host in NSW, Victoria (VIC), WA and SA; and A. tenuissima on B. napus in NSW, SA and WA. It is evident that alternaria leaf spot on canola across southern Australia is not solely caused by A. brassicae, but that a range of other Alternaria spp. are also involved to varying degrees, depending upon the year and the geographic locality.     

Pathogenic variation of Alternaria species associated with leaf blotch and fruit spot of apple in Australia

Four Alternaria species groups (A. longipes, A. arborescens, A. alternata/A. tenuissima and A. tenuissima/A. mali) are associated with leaf blotch and fruit spot of apple in Australia. There is no information on the variability of pathogenicity among the species and isolates within each species causing leaf blotch or fruit spot. We used a detached leaf assay and an in planta fruit inoculation assay to determine the pathogenicity and virulence of the four Alternaria species. Our results showed that isolates within the same species were not specific to either leaf or fruit tissue and showed great variability in pathogenicity and virulence, indicating cross-pathogenicity, which may be isolate dependent rather than species dependent. Generally, virulence of A. tenuissima and A. alternata isolates on leaf and fruit was higher than other species. Isolates of all species groups were pathogenic on leaves of different cultivars, but pathogenicity on fruit of different cultivars varied among isolates and species. Implications of our findings on prevalence of the diseases in different apple-producing regions in Australia and the development of targeted disease management of the diseases are discussed.     

Species prevalence and disease progression studies demonstrate a seasonal shift in the Alternaria population composition on potato

To assess the incidence of early blight/brown spot (EB/BS) in Flanders (Belgium), potato fields in 22 locations were monitored and scored over the duration of the growing seasons 2014 and 2015. The average disease incidence was shown to be higher in 2014 than in 2015. Soil type, rainfall and temperature were also analysed in relation to disease incidence. In 2014, potato plants grown in sandy soils had more EB/BS disease than those grown in clay or loamy soils. However, the low disease incidence in 2015 meant that differences in disease levels between soil types could not be discerned for that growing season. A windowpane analysis demonstrated that rainfall and humidity accounted for the differences in disease incidence between the two growing seasons. During the course of the survey, the species composition in leaves with symptoms was assessed using real‐time PCR. Remarkably, small‐spored Alternaria species, such as A. alternata and A. arborescens, rather than the more virulent A. solani were the predominant species on potato leaves throughout the growing season. As the disease progressed, the proportion of A. solani increased. In view of these results, the virulence of a collected set of Alternaria isolates was assessed by an in vitro assay. Despite A. solani being more virulent than A. alternata or A. arborescens, the most abundant species isolated from potato leaves with symptoms was A. arborescens.     

Characterization of Alternaria species associated with leaf blight of sunflower in China

Nine Alternaria species have been reported to be associated with sunflower leaf blight worldwide, and A. helianthi has been recognized as the most prevalent and damaging species. However, the population structure of Alternaria species causing leaf blight of sunflower in China had not been examined thoroughly prior to this study. During 2010 to 2013, a total of 272 Alternaria isolates were obtained from infected sunflower leaves in 11 provinces, autonomous regions, and municipalities of China. Based on morphological traits and sequence analyses of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) and the partial coding sequences of the histone 3 gene, 227 (83.5 %) isolates were identified as belonging to Alternaria tenuissima, the remainder 45 isolates were grouped to A. alternata (16.5 %). Compared with the ITS regions of rDNA, sequence analyses of the partial coding sequences of histone 3 gene displayed a critical role in discrimination of the small-spored Alternaria species. Phylogenetic analysis of the partial coding sequence of histone 3 gene clearly divided the representative Alternaria isolates into two main clades, A. tenuissima and A. alternata. The pathogenicity of A. tenuissima and A. alternata on detached leaves of sunflower cv. Gankui No.2 did not significantly differ between the two species or among isolates from different geographical origins. Our results indicate that the population structure of Alternaria species associated with sunflower leaf blight differed from that reported previously in China since A. helianthi was not found in this study. In addition, this is the first report about A. tenuissima causing leaf blight on sunflower in China.     

Pathogenicity,Morpho-Species and Mating Types of <Emphasis Type="Italic">Alternaria</Emphasis> spp. causing Alternaria blight in <Emphasis Type="Italic">Pistacia</Emphasis> spp. in Turkey

Alternaria genus includes many plant pathogens on numerous hosts, causing leaf spots, rots and blights. Alternaria blight has been observed as one of the important fungal diseases of pistachio (Pistacia vera L.) as well as its wild relatives (P. terebinthus, P. lentiscus, P. khinjuk, P. atlantica, P. mutica) in Turkey. Alternaria species were sampled from Pistacia spp. hosts from different geographic regions in Turkey during field trips in late spring to early fall of 2013. Alternaria blight symptoms were observed mainly on fruits and rarely on leaves. Four hundred and twenty two of the isolates were morphologically defined as A. alternata, A. tenuissima, A. arborescens and also intermediate morpho-species between A. alternata/A. arborescens. Pathogenicity of the isolates was confirmed with host inoculations on detached fruits. Mating types of 270 isolates of Alternaria spp. from the collection were identified using a PCR-based mating type assay that amplifies either a MAT1-1 or a MAT1-2 fragment from the mating locus. Although a strongly clonal population structure was expected due to the putative asexual reproduction of these fungi, both idiomorphs were detected at equal frequencies at several different spatial scales. The distribution of mating types within each geographic region, within host species as well as in overall collection was not significantly different from 1:1. Amplified fragments of partial idiomorph sequences were obtained for representative isolates. Parsimony trees were depicted based on sequence data of mating type genes for these representative isolates as well as some other Alternaria species obtained by Genebank. Several point mutations presented a few clusters which are supported by high bootsrapped values. The Alternaria blight disease agents both from cultivated and wild hosts were pathogenic on pistachio which may cause difficulties to control the disease because of extensity of pathogen sources. Besides, equal mating type distribution of the pathogen at both geographic and host species levels suggests a potential for sexual reproduction of Alternaria spp. in Turkey.     

Resistance to azoxystrobin in Alternaria isolates from pistachio in California

Failure to control Alternaria late blight in a few California pistachio orchards was observed after only 3-4 years of consecutive applications of azoxystrobin-based fungicide programs. A total of 72 isolates of Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens, the causal organisms of Alternaria late blight, were collected from pistachio orchards with (58 isolates) and without (14 isolates) a prior history of azoxystrobin applications. The sensitivity to azoxystrobin was determined in conidial germination assays. Isolates from orchards with a history of azoxystrobin applications had EC₅₀ values greater than 100 μg/ml, whereas isolates from orchards without a prior history of azoxystrobin usage had EC₅₀ values ranging from 0.008 to 0.045 μg/ml. Azoxystrobin resistance correlated with a single mutation in the cytochrome b (cyt b) gene causing a change of glycine to alanine at amino acid position 143. A pair of PCR primers AF and AR was developed that amplified a 226-bp DNA fragment of the cyt b gene containing the mutation site from all three Alternaria species but not from 30 other fungal species frequently found on pistachio. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction enzyme Fnu4HI allowed differentiation of the PCR fragment of wild type cyt b gene from that of mutated gene. This method will aid in a fast detection of azoxystrobin resistance in these three Alternaria species.     

Characterization of iprodione-resistant Alternaria isolates from pistachio in California

Alternaria late blight caused by Alternaria spp. in the alternata, tenuissima, and arborescens species-groups is one of the most common fungal diseases of pistachio in California. In this study, a field iprodione-resistant (FIR) isolate of the arborescens species-group and a laboratory-induced iprodione-resistant (LIIR) isolate of the alternata species-group were characterized by fungicide and osmotic sensitivity, virulence on detached pistachio leaflets, and sequence of the coiled-coil region (six repeats of approximately 90-amino-acid domain) of the two-component histidine kinase (HK) gene. Both FIR and LIIR isolates were sensitive to azoxystrobin and tebuconazole, and azoxystrobin-resistant isolates were sensitive to iprodione and tebuconazole. The LIIR isolate showed more sensitivity to osmotic stress than its wild-type parent. However, the FIR isolate did not show higher osmotic sensitivity compared to field iprodione-sensitive (FIS) isolates. Laboratory inoculation tests showed that both FIR and LIIR isolates remained highly virulent on pistachio. Analysis of DNA sequences of the HK coiled-coil region showed that there were no differences in deduced amino acid sequence of this region from the LIIR, FIR, and FIS Alternaria isolates from pistachio in California.     

Evaluating aggressiveness and host range of Alternaria dauci in a controlled environment

The aggressiveness of Alternaria dauci isolates was investigated in greenhouse conditions. Twenty‐seven isolates were pre‐selected from a large collection to represent high diversity according to geographic or host origins and intergenic spacer (IGS) polymorphism. IGS sequence analysis revealed that isolates were grouped within three different clusters. Eleven isolates were selected and inoculated on a susceptible carrot cultivar. Three criteria (mean lesion number, mean necrotic leaf area and mean disease index) were used to assess the aggressiveness of isolates. Continuous variation in aggressiveness was shown and no clear division into isolate classes was evident. For the host range study, two isolates were inoculated under greenhouse conditions onto nine cultivated Apiaceae species, two wild Daucus species and six cultivated non‐Apiaceae species representing six botanical families. Lesions varying in severity were observed on all dicot species (Apiaceae and non‐Apiaceae), but no symptoms developed on the two monocots studied (leek and sweetcorn). Plant species were also differentiated on the basis of expanding lesions (cultivated and wild carrot, dill and fennel) or non‐expanding lesions (other dicot species). Typical A. dauci conidia were observed after in vitro incubation of leaves with symptoms. Fungal structures were isolated from lesions and A. dauci was confirmed on the basis of conidial morphology and specific conventional PCR results. Genotyping of individual isolates performed with microsatellite markers confirmed the presence of the inoculated isolate. The results clearly showed that, in controlled conditions, the host range of A. dauci is not restricted to carrot.     

Variability of Cuban and International Populations of Alternaria solani from Different Hosts and Localities: AFLP Genetic Analysis

As causal agent of early blight disease in tomato and potato, Alternaria solani is an internationally important horticultural pathogen. Genetic variability was surveyed by amplified fragment length polymorphism analysis in a total of 112 isolates from potato and tomato, representing pathogen populations from different Cuban provinces together with isolates from the USA, Brazil, Turkey, Greece and China. Also included in the analysis were isolates from catenulated Alternaria spp. from Brazil, Canada, Greece and Russia, along with single isolates of Alternaria porri, Alternaria alternata and a Curvularia sp. UPGMA clustering revealed a differentiation between the isolates of A. solani and all other species with the exception of A. porri which could not be distinguished from A. solani. Among the isolates of A. solani, two distinct subclusters were formed, with high genetic significance revealed by bootstrapping, corresponding to a general subdivision based on the respective solanaceous host. The results are discussed with regard to potential host specificity of A. solani on tomato and potato, and in terms of the comparative contributions of regional and international genetic variability in populations of this ubiquitous plant pathogen.     

Ethylene signalling is essential for the resistance of Nicotiana attenuata against Alternaria alternata and phytoalexin scopoletin biosynthesis

The phytohormone ethylene plays an important role in plant defence responses to pathogen attack. When infected by the necrotrophic fungal pathogen Alternaria alternata (tobacco pathotype), which causes severe diseases in Nicotiana species, the wild tobacco plant Nicotiana attenuata accumulates a high amount of the jasmonate (JA)‐dependent phytoalexin scopoletin to defend itself against this fungal pathogen. However, it is still not known whether ethylene signalling is also involved in scopoletin biosynthesis and the resistance of N. attenuata. After infection, ethylene biosynthetic genes were highly elicited. Furthermore, plants strongly impaired in ethylene biosynthesis or perception had dramatically decreased scopoletin levels, and these plants became more susceptible to the fungus, while A. alternata‐elicited JA levels were increased, indicating that the decreased defence responses were not due to lower JA levels. Thus, it is concluded that after infection, ethylene signalling is activated together with JA signalling in N. attenuata plants and this subsequently regulates scopoletin biosynthesis and plant resistance.     

Black Spot of Peach Caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler

A new disease of peach (Prunus persica Batsch var. vulgaris Maxim.), causing brown, sunken lesions and brownish to blackish brown spots with cracks on peach fruits, was found in Okayama prefecture, Japan, in 1995. The disease was observed not only on peach fruits but also on twigs and leaves. An Alternaria sp. was consistently isolated from these diseased fruits, twigs and leaves. The isolates were pathogenic to peach fruits and leaves. Based on the morphological characteristics, the causal fungus was identified as Alternaria alternata (Fr.) Keissler. After cross-inoculation with isolates from peach, Japanese pear and apple, the isolates were found to be pathogenic only to their original host. This is the first report on a peach disease caused by a host-specific A. alternata; therefore, the common name of black spot (`Kokuhanbyo' in Japanese) was proposed. Received 25 June 1999/ Accepted in revised form 12 October 1999     

Control of infection of tomato fruits by <Emphasis Type="Italic">Alternaria</Emphasis> and mycotoxin production using plant extracts

Tomato fruits are susceptible to infection by Alternaria species. In addition, Alternaria species may contaminate the fruits with mycotoxins. There is thus interest in control systems to minimise pathogenicity and control toxin production. The objectives of this study were to examine the effect of plant extracts of Eucalyptus globulus and Calendula officinalis on the growth of strains of Alternaria alternata and A. arborescens, on pathogenicity of tomato fruits and mycotoxin production. The growth bioassays showed that the ethanolic and chloroformic fractions of E. globulus were the most effective in reducing growth of A. alternata (66–74 %) and A. arborescens (86–88 %), respectively at 2500 μg/g. The effects of plant extracts on mycotoxin biosynthesis were variable and strain dependent. The most effective fractions in decreasing mycotoxin accumulation were the ethanolic and chloroformic extracts of E. globulus, which reduced tenuazonic acid by 89 %, alternariol by 75–94 % and almost complete inhibition of alternariol monomethyl ether. All the tested fractions reduced percentage of infected tomato fruits when compared to the controls. The ethanolic and chloroformic fractions of E. globulus completely inhibited growth of A. alternata and A. arborescens on unwounded fruits and reduced the aggressiveness on wounded fruits of strains of both species significantly.     

Pathogenicity and toxigenicity of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> isolates collected from maize roots,stems and ears in South Africa

Sunflower (Helianthus annuus L.) is an important oilseed crop in South Africa, and is grown in rotation with maize in some parts of North West, Limpopo, Free State, Mpumalanga and Gauteng provinces. Alternaria leaf blight is currently one of the major potential disease threats of sunflower and is capable of causing yield losses in all production regions. Alternaria helianthi was reported as the main cause of Alternaria leaf blight of sunflower in South Africa; however small-spored Alternaria species have been consistently isolated from leaf blight symptoms during recent surveys. The aim of this study was to use morphological and molecular techniques to identify the causal agent(s) of Alternaria blight isolated from South African sunflower production areas. Alternaria helianthi was not recovered from any of the sunflower lesions or seeds, with only Alternaria alternata retrieved from the symptomatic tissue. Molecular identification based on a combined phylogenetic dataset using the partial internal transcribed spacer regions, RNA polymerase second largest subunit, glyceraldehyde-3-phosphate dehydrogenase, translation elongation factor and Alternaria allergen gene regions was done to support the morphological identification based on the three-dimensional sporulation patterns of Alternaria. Furthermore, this study aimed at evaluating the pathogenicity of the recovered Alternaria isolates and their potential as causal agents of Alternaria leaf blight of sunflower. Pathogenicity tests showed that all the Alternaria alternata isolates tested were capable of causing Alternaria leaf blight of sunflower as seen in the field. This is the first report of A. alternata causing leaf blight of sunflower in South Africa.     

Evaluation of virulence and pathogenicity of Alternaria patula on French marigold (Tagetes patula)

Alternaria patula, the cause of French marigold leaf black spot and flower blight, was first isolated from seeds of French marigold cv. Queen Sophia. It is described as a new species of Alternaria and has a considerable morphological variation with a preferential pathogenicity to Asteraceae, Solanaceae and Cucurbitaceae plants. Alternaria patula produces an array of pectin depolymerases that can break 1,4-α-glycosidic bonds either by hydrolysis of polygalacturonases (PG, E.C. 3.2.1.15) or via trans-elimination of pectate lyases (PL, E.C. 4.2.2.2) and pectin lyases (PNL, E.C. 4.2.2.10). This study is the first to emphasize the variability and significance to pathogenesis of the pectinolytic enzymes of A. patula that target various pectic polymers structures during host tissue invasion. Alternaria patula also produced zinniol derivatives as non-host-specific toxins (nHSTs), albeit without phytotoxic symptoms in French marigold. The management of Alternaria blight caused by A. patula via the application of pyrifenox and antagonistic Bacillus amyloliquefaciens effectively reduced disease severity, without adverse effects on French marigold in both in vitro and in vivo bioassays.     

Identification and characterization of <Emphasis Type="Italic">Alternaria</Emphasis> species causing leaf spot on cabbage,cauliflower, wild and cultivated rocket by using molecular and morphological features and mycotoxin production

Alternaria species are common pathogens of fruit and vegetables able to produce secondary metabolites potentially affecting human health. Twenty-nine isolates obtained from cabbage, cauliflower, wild and cultivated rocket were characterized and identified based on sporulation pattern and virulence; the phylogenetic analysis was based on the β-tubulin gene. Isolates were identified as A. alternata, A. tenuissima, A. arborescens, A. brassicicola and A. japonica. Pathogenicity was evaluated on plants under greenhouse conditions. Two isolates showed low level of virulence on cultivated rocket while the other isolates showed medium or high level of virulence. Isolates were also characterized for their mycotoxin production on a modified Czapek-Dox medium. Production of the five Alternaria toxins, tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene and tentoxin were evaluated. Under these conditions, about 80% of the isolates showed the ability to produce at least one mycotoxin.     

Population structure and genetic diversity suggest recent introductions of Dothistroma pini in Slovakia

Dothistroma pini is one of two pathogens causing Dothistroma needle blight (DNB), a foliar disease of pines. The species was redefined in 2004 and subsequently recorded in several European countries. In Slovakia, the first report of the pathogen was in 2013. In this study, the population structure, genetic diversity, and reproductive mode of 105 isolates collected from 10 localities and seven hosts were determined in Slovakia. Species-specific mating type markers, ITS haplotype determination, and 16 microsatellite markers were used to characterize and genotype the isolates. Overall, 15 unique multilocus haplotypes (MLHs) based on microsatellite markers and three ITS haplotypes were identified. Three independent methods (DAPC, STRUCTURE, EDENetwork) separated the isolates into two distinct population clusters corresponding with ITS haplotypes. A high level of clonality was recorded suggesting that conidia are the primary source of pathogen dispersal. The low genetic diversity, predominantly asexual reproductive mode of the pathogen, and the fact that most isolates were collected from introduced tree species and native species in artificially planted urban greenery, supports the hypothesis that D. pini has been recently introduced into Slovakia.     

Sensitivity of Red Delicious Apple Fruit at Various Phenologic Stages to Infection by Alternaria alternata and Moldy-core Control

The fungus Alternaria alternata, is considered to be the predominant fungus involved in moldy-core of Red Delicious strains of apple. In this paper, we report on the sensitivity of various phenologic stages to infection by A. alternata, and on the efficacy of various fungicides in controlling moldy-core disease in apple orchards. Artificial inoculations conducted in the orchard during 1999 and 2000 seasons revealed that the beginning of bloom (10–30%) and full bloom were the most susceptible developmental stages for infection. Natural infection with A. alternata in fruits was relatively high, reaching 44% and 46% of the fruits on control non-treated trees in 1999 and 2000. Four foliar applications of polyoxin B, difenoconazole and azoxystrobin, starting from the beginning of bloom until fruit set, reduced the number of infected fruits by 54–70%, 61–70% and 50–65%, respectively, compared with non-treated trees. Four or eight foliar applications of each fungicide provided similar levels of control. There were no significant differences between two, four or six foliar applications of difenoconazole, neither between two or four applications of polyoxin B. Adding CaCl₂, as a tank mixture with difenoconazole at full rate, did not improve efficacy. Alternaria was recovered from the inner part of the core region of 71–88% of the fruits of the non-treated control, but was recovered less frequently from the outside part of the core region. Fruits of difenoconazole and polyoxin B treated trees were less colonized with A. alternata at both the inner and outside parts of the core region, as compared with controls. Results indicate that a control programme based on spray applications of difenoconazole or polyoxin B, during bloom period, can effectively reduce Alternaria on Red Delicious.