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.     

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.     

Contrasting species boundaries between sections Alternaria and Porri of the genus Alternaria

The fungal genus Alternaria comprises a large number of asexual taxa with diverse ecological, morphological and biological modes ranging from saprophytes to plant pathogens. Understanding the speciation processes affecting asexual fungi is important for estimating biological diversity, which in turn affects plant disease management and quarantine enforcement. This study included 106 isolates of Alternaria representing five phylogenetically defined clades in two sister sub‐generic groups: section Porri (A. dauci, A. solani and A. limicola) and section Alternaria (A. alternata/tenuissima and A. arborescens). Species in section Porri are host‐specific while species in section Alternaria have wider host ranges. For each isolate, DNA sequences of three genes (Alt a1, ATPase, Calmodulin) were used to estimate phylogenies at the population and species levels. Three multilocus haplotypes were distinguished among A. dauci isolates and only one haplotype among A. solani and A. limicola isolates, revealing low or no differentiation within each taxon and strong clonal structure for taxa in this section. In contrast, 37 multilocus haplotypes were found among A. alternata/tenuissima isolates and 21 multilocus haplotypes among A. arborescens isolates, revealing much higher genotypic diversity and multiple clonal lineages within taxa, which is not typical of asexual reproducing lineages. A species tree was inferred using a Yule Speciation model and a strict molecular clock assumption. Species boundaries were well defined within section Porri. However, species boundaries within section Alternaria were only partially resolved with no well‐defined species boundaries, possibly due to incomplete lineage sorting. Significant association with host specificity seems a driving force for speciation.     

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.     

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.     

Characterization of <Emphasis Type="Italic">Phaeoacremonium</Emphasis> isolates associated with Petri disease of table grape in Northeastern Brazil,with description of <Emphasis Type="Italic">Phaeoacremonium nordesticola</Emphasis> sp. nov.

Severe outbreaks of Alternaria leaf blotch and fruit spot were recently observed in cv. Pink Lady apples in northern Israel, especially on fruit. Such severe outbreaks have not been reported from other countries. Symptoms involved cracks and rot around the calyx and external rot of the fruit body. Up to 80 % of the fruit in some orchards were affected by the disease. Microscopic examinations, fulfillment of Koch’s postulates and molecular (genetic) analyses confirmed the causal agent as Alternaria alternata f. sp. mali. The incidence of Alternaria increased as the degree of calyx cracking increased, or if fruit were both cracked and rotted. Injecting spore suspensions into the fruit produced typical rot symptoms. Injection assays of detached fruit of eight apple cultivars showed that cvs. Pink Lady and Golden Delicious were susceptible whereas cv. Jonathan was resistant. Pink Lady and Golden Delicious produced more fruit rot as the inoculum concentration increased. Rot in all three cultivars was moderate close to the skin but more severe close to the seed locule. Aqueous extracts taken from Jonathan fruit peel inhibited germ tube elongation of A. alternata f. sp. mali in vitro. This is the first report on heavy infection of Pink Lady fruit in Israel caused by A. alternata f. sp. mali.     

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.     

Detection of Thiophanate-methyl-resistant Strains in Diplocarpon mail, Causal Fungus of Apple Blotch

Isolates of Diplocarpon mali, causal fungus of apple blotch, collected from four prefectures in Japan in 1997–1998 were tested for sensitivity to thiophanate-methyl. Results from mycelial growth tests showed that MIC values of the fungicide were 0.19 μg/ml against all isolates from Akita, Nagano and Saga prefectures but 100 or 200 μg/ml against all isolates from the Tokusa area in Yamaguchi prefecture. Detached apple leaves sprayed with the fungicide developed severe symptoms when inoculated with the isolate from Tokusa, but developed no symptoms with the isolate from Nagano. These results are the first confirmation of thiophanate-methyl-resistant strains in D. mali. Received 1 September 1999/ Accepted in revised form 15 October 1999     

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.     

云南蓝莓叶斑病的病原菌鉴定

<正>蓝莓(Vaccinium spp.)属于杜鹃花科(Ericaceae)越橘属(Vaccinium)多年生小浆果类果树,广泛应用于医药、保健、化妆品和环境保护等领域,联合国粮农组织将其果实列为世界五大健康食品之一。我国已有10多个省份开始大面积的蓝莓商业化栽培[1],这也是南方酸性土丘陵地区值得发展的经济作物。随着栽培面积的不断扩大,蓝莓病害日趋突出,严重影响和制约蓝莓产业的发展。     

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     

Characterization of Neofabraea vagabunda isolates causing apple bull's eye rot in Italy (Emilia‐Romagna region)

Neofabraea vagabunda is the prevalent cause of bull's eye rot, one of the main postharvest diseases of apple, in many producing areas, but its biology has not been studied in detail. The molecular identification, by DNA sequencing of the β‐tubulin region, of 41 isolates collected from apples showing bull's eye rot in the Emilia‐Romagna region confirmed N. vagabunda as the main species in Italy. A biological and morphological characterization of N. vagabunda isolates was performed in vitro. Assays at temperatures ranging from 0 to 30 °C carried out on 10 isolates demonstrated: (i) a marked influence of temperature on colony morphology, conidial production, conidial size and mycelial growth, showing the cold‐tolerant character of N. vagabunda; and (ii) that culture at 15 °C on tomato agar (TA) for 14 days is a rapid and reliable method to favour pathogen conidial production. Trials performed on 38 isolates using these incubation conditions recorded the presence of two N. vagabunda morphotypes, differing for colony morphology, conidial size, conidiomata formation and temperature requirement. The alkalizing ability of the pathogen during growth on TA was also demonstrated for the first time. The pathogenicity of 25 N. vagabunda isolates was proved in vivo on artificially infected Cripps Pink apples. A pH increase was also recorded in apple tissue infected by N. vagabunda isolates (on average 0.2 and 0.3 units of pH after 60 and 120 days of incubation, respectively), suggesting that the N. vagabunda transition from quiescence to necrotrophic colonization in apples could involve the secretion of alkalizing compounds.     

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.     

Morphological and molecular diversity of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily‐primed PCR (ap‐PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap‐PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap‐PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β‐tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap‐PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.     

A rapid virulence assay for the Dutch elm disease fungus Ophiostoma novo‐ulmi by inoculation of apple (Malus × domestica ‘Golden Delicious’) fruits

Large‐scale virulence tests using trees or saplings are expensive, time‐consuming and require a considerable amount of space. The suitability of using ‘Golden Delicious’ apples as a rapid screen for identifying Ophiostoma novo‐ulmi transformants with reduced virulence was thus evaluated. When a collection of O. novo‐ulmi field isolates belonging to subspecies novo‐ulmi or americana was inoculated to apples, members of subsp. novo‐ulmi induced, on average, larger necrotic lesions than subsp. americana isolates. The size of the lesions on apples was not correlated with mycelial growth rate of isolates on nutrient agar. Insertional mutants from O. novo‐ulmi subsp. novo‐ulmi isolate H327 were inoculated to ‘Golden Delicious’ apples and Ulmus parvifolia × U. americana saplings in parallel experiments. Results clearly indicated that the O. novo‐ulmi transformants included several exhibiting significantly altered levels of virulence. Variability among replicates within a treatment was reduced in apple inoculation data compared to elm sapling data. Overall, the ‘Golden Delicious’ apple assay was found to be an excellent means for rapidly assessing the virulence level of O. novo‐ulmi isolates.     

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.     

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.     

Suppression of seed-borne Alternaria arborescens and growth enhancement of wheat with biorational fungicides

Alternaria spp. are among the major fungal contaminants of wheat grain under postharvest and storage conditions, where A. arborescens was recently detected as a new member of this complex in Argentina causing black point. The aim of this study was to assess the potential of some biorational agents to control A. arborescens and their plant growth promoting of wheat. Seed treatments with spore suspensions of Trichoderma harzianum and Eppicoccum nigrum, extracts from Lippia alba and garlic, sodium bicarbonate, salicylic acid (SA), potassium chloride and dibasic sodium phosphate (SP) were applied to grains of wheat cultivar BIOINTA 1004 before their inoculation with the pathogen. After 7 days, seed germination and infection, necrotic symptoms on emerged seedlings and fresh weight were evaluated. Remarkable results were obtained with L. alba, SA and SP treatments that reduced symptoms markedly compared with the control. Interestingly, necrosis of radicles was significantly reduced by the application of all treatments tested. Moreover, fresh weight of seedlings was significantly increased with the application of the two antagonists, diluted garlic juice and the three tested salts in comparison with controls. Therefore, a positive role as growth promoters can be elucidated. It is concluded that compounds here tested have potential as ecofriendly alternatives to control seed-borne Alternaria fungi of wheat.     

The threat of wild habitat to scab resistant apple cultivars

Evaluations of plant resistance to pathogens are rarely made using isolates from wild habitats, although the heterogeneity of such habitats may generate pathogen diversity which could be a source of new virulence in cultivated habitats. The aim of this study was to investigate whether scab resistance factors, identified and characterized in apples using isolates of Venturia inaequalis from a cultivated habitat, remained effective against isolates from a wild habitat. Three V. inaequalis core collections originating from the cultivated apple Malus × domestica and from two wild species, M. sieversii and M. sylvestris, were established to maximize pathogen diversity. For each core collection, 10 isolates were inoculated in mixtures onto 51 genotypes from an apple progeny segregating for two qualitative resistance genes and six quantitative resistance loci (QRL). On each apple genotype, isolates that contributed to the scab symptoms were identified within the mixture using microsatellite markers. The most frequently detected isolates were inoculated singly to compare their aggressiveness according to their host origin. The results showed that isolates from a wild habitat were able to infect the susceptible apple genotypes. However, these isolates were never more aggressive than isolates from the cultivated habitat on the resistance factors tested. It can therefore be concluded that the resistance factors used in this study, identified with V. inaequalis isolates from a cultivated habitat, remained effective against isolates from M. sylvestris and M. sieversii.