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.     

Characterization of Colletotrichum orchidophilum,the agent of black spot disease of vanilla

Black spot symptoms were reported on vanilla plants in Reunion Island in 2011. They have repeatedly reduced annual pod production by 10% to 30%. The disease is characterized by dark spots that appear in slight depressions on flowers, pods, leaves and stems. The spots then develop into broad clearly depressed necrotic plaques. Morphological and molecular analyses, as well as pathogenicity tests, identified the fungus Colletotrichum orchidophilum (Ascomycota) as the causal agent of the disease. Inoculation tests in controlled conditions confirmed that the two C. orchidophilum strains isolated from fruit lesions are pathogenic on the leaves and fruits of Vanilla planifolia (accessions CR0001 and CR0020). However, these strains induced symptoms only when the epidermis of leaves and fruits had been punctured by a needle. In the absence of injury, no symptom appeared. Colletotrichum arxii and Fusarium proliferatum (Ascomycota) are fungal species that are also frequently isolated from black spot lesions. However, they are not pathogenic to vanilla. This is the first report of C. orchidophilum in Reunion Island. It is also the first demonstration of C. orchidophilum's pathogenicity to an orchid. Simple preventive control measures were proposed to reduce the incidence of black spot disease in vanilla plots.     

Taxonomic Characterization and Experimental Host Ranges of Four Newly Recorded Species of Alternaria from Japan

Alternaria fungi are important plant pathogens. Here, we identified three species new to the Japanese mycoflora: Alternaria celosiae, Alternaria crassa and Alternaria petroselini. We proposed a new name for A. celosiae (E.G. Simmons & Holcomb) Lawrence, Park & Pryor, a later homonym of A. celosiae (Tassi) O. S?vul. To characterize these and a fourth morphological taxon, Alternaria alstroemeriae, which was recently added to Japan's mycoflora, an integrated species concept was tested. We determined the host range of each isolate using inoculation tests and analysed its phylogenetic position using sequences of the internal transcribed spacer rDNA. The pathogenicity of our A. alstroemeriae isolate was strictly limited to Alstroemeria sp. (Alstroemeriaceae), but the species was phylogenetically indistinguishable from other small‐spored Alternaria. Alternaria celosiae on Celosia argentea var. plumosa (Amaranthaceae) was also pathogenic to Amaranthus tricolor, to Alternanthera paronychioides and weakly to Gomphrena globosa (all Amaranthaceae) and formed a clade with the former Nimbya celosiae. Alternaria crassa on Datura stramonium (Solanaceae) was also pathogenic to Brugmansia × candida and Capsicum annuum in Solanaceae, but not to other confamilial plants; phylogenetically it belonged to a clade of large‐spored species with filamentous beaks. Morphological similarity, phylogenetic relationship and experimental host range suggested that A. crassa, Alternaria capsici and Alternaria daturicola were conspecific. Alternaria petroselini on Petroselinum crispum (Apiaceae) was pathogenic to five species in the tribe Apieae as well as representatives of Bupleureae, Coriandreae, Seliaeae and Scandiceae in Apiaceae. Both phylogeny and morphology suggested conspecificity between A. petroselini and Alternaria selini.     

First Report of Pseudomonas lurida Causing Bacterial Leaf Spot on Miscanthus sinensis

Miscanthus spp. are large perennial wetland grasses that are receiving considerable attention as bioenergy crops. In late summer 2011, leaf spot symptoms were observed in a field of Miscanthus sinensis in Jeongseon, Gangwon province, Korea. Bacterial strains that belonged to the γ‐Proteobacteria genus Pseudomonas were isolated from the affected leaves. By phylogenetic analysis and phenotypic characterization, the representative strain MDM‐03 was identified as Pseudomonas lurida. Healthy M. sinensis leaves inoculated with MDM‐03 developed leaf spots similar to those observed in field. Bacteria re‐isolated from the leaf lesions were identical to the original strain MDM‐03 based on their cultural characteristics and 16S rDNA sequencing. This is the first report of bacterial leaf spot in Miscanthus sinensis.     

Occurrence and Characterization of the Bacterial Spot Pathogen Xanthomonas euvesicatoria on Pepper in Iran

We report in this study for the first time the occurrence of bacterial spot of pepper in Iran and both phenotypic and genetic characterization of its causal agent, Xanthomonas euvesicatoria. Pepper plants grown in 15 of 30 surveyed private gardens and commercial fields were infected by the pathogen in Marand County, East Azerbaijan Province, north‐western Iran. The obtained strains of X. euvesicatoria had different amylolytic and pectolytic activities compared with those reported for this species elsewhere. Pathogenicity tests showed that strains isolated from diseased pepper are able to infect tomato, in addition to pepper. Host range of the pathogen was assessed on eight annual plant species including crops and weeds by measuring the population dynamics. The host range assessment showed that in addition to pepper and tomato, known hosts of X. euvesicatoria, the Iranian strains were able to colonize a number of new hosts such as nightshade and common bean. In contrast, none of them were able to build up their population on cowpea, eggplant, bindweed and zucchini. All X. euvesicatoria strains obtained in this study were sensitive to copper sulphate and streptomycin at concentrations higher than 20 and 50 mg/l, respectively. Phylogenetic analyses of the strains using the sequences of gyrB and hrpB genes confirmed their species as X. euvesicatoria. Given a direct commercial trade of fresh solanaceous vegetables between Iran and Turkey, it is hypothesized that the pathogen entered north‐western Iran from eastern parts of Turkey through infected plant materials. Finally, the role of prevention – based on the use of healthy planting materials and resistant and/or tolerant plant varieties – to contain the potential disease epidemics is discussed.     

Identification of Alternaria tenuissima causing brown leaf spot on Paris polyphylla var. chinensis in China

Severe brown leaf spot disease was observed on Paris polyphylla var. chinensis in Sichuan Province, China, in 2017 and 2018. The initial symptoms were many light‐brown small spots with necrotic centres, round or irregular in shape, becoming dark brown, gradually enlarging and eventually coalescing, causing extensive leaf senescence. A fungus was isolated from diseased leaves showing typical symptoms of brown leaf spot. The isolates were cultured on potato sucrose agar, and their morphological characteristics of the causal pathogen were observed under a light microscope. Pathogenicity tests revealed that this fungus was the causal pathogen of the disease. Molecular analyses of the sequences of the ribosomal DNA internal transcribed spacer (ITS) region, translation elongation factor 1‐alpha (TEF) and the RNA polymerase II second largest subunit (RBP2) gene were conducted to confirm the identity of the pathogen. The multi‐gene phylogeny indicated that the causal agent was Alternaria tenuissima. To our knowledge, this is the first report of A. tenuissima causing brown leaf sports on P. polyphylla var. chinensis in China.     

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.     

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.     

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.     

Identification and Molecular Characterizations of Neoscytalidium dimidiatum Causing Stem Canker of Red‐fleshed Dragon Fruit (Hylocereus polyrhizus) in Malaysia

During the year 2008 to 2009, a new disease of stem canker was noticed in most red‐fleshed dragon fruit (Hylocereus polyrhizus) plantations in Malaysia. The symptoms observed were small circular sunken orange spot, black pycnidia and rotted stem. This study was conducted to determine the occurrence of the stem canker on H. polyrhizus in Malaysia, subsequently to isolate, identify and characterize the fungal pathogen based on morphology and molecular characteristics and pathogenicity test. From the surveyed 20 plantations in Malaysia, stem canker was detected in all the plantations. A total of 40 isolates of Scytalidium‐like fungus were isolated and identified as Neoscytalidium dimidiatum based on morphological characteristics and ITS region sequences, which showed 99% similarity to N. dimidiatum (FJ648577). From the phylogenetic analysis using maximum‐likelihood tree, isolates of N. dimidiatum from stem canker of H. polyrhizus were grouped together and did not show any sequence variation. From pathogenicity test, all 40 isolates of N. dimidiatum were pathogenic causing stem canker on H. polyrhizus. To our knowledge, this is the first report of stem canker of H. polyrhizus caused by N. dimidiatum in Malaysia.     

Overexpression of FBR41 enhances resistance to sphinganine analog mycotoxin‐induced cell death and Alternaria stem canker in tomato

Fumonisin B1 (FB1) and Alternaria alternate f. sp. lycopersici (AAL)‐toxin are classified as sphinganine analog mycotoxins (SAMTs), which induce programmed cell death (PCD) in plants and pose health threat to humans who consume the contaminated crop products. Herein, Fumonisin B1 Resistant41 (FBR41), a dominant mutant allele, was identified by map‐based cloning of Arabidopsis FB1‐resistant mutant fbr41, then ectopically expressed in AAL‐toxin sensitive tomato (Solanum lycopersicum) cultivar. FBR41‐overexpressing tomato plants exhibited less severe cell death phenotype upon AAL‐toxin treatment. Analysis of free sphingoid bases showed that both fbr41 and FBR41‐overexpressing tomato plants accumulated less sphinganine and phytosphingosine upon FB1 and AAL‐toxin treatment, respectively. Alternaria stem canker is a disease caused by AAL and responsible for severe economic losses in tomato production, and FBR41‐overexpressing tomato plants exhibited enhanced resistance to AAL with decreased fungal biomass and less cell death, which was accompanied by attenuated accumulation of free sphingoid bases and jasmonate (JA). Taken together, our results indicate that FBR41 is potential in inhibiting SAMT‐induced PCD and controlling Alternaria stem canker in tomato.     

Molecular Identification,Mycotoxin Production and Comparative Pathogenicity of Fusarium temperatum Isolated from Maize in China

A recently isolated Fusarium population from maize in Belgium was identified as a new species, Fusarium temperatum. From a survey of Fusarium species associated with maize ear rot in nineteen provinces in 2009 in China, ten strains isolated from Guizhou and Hubei provinces were identified as F. temperatum. Morphological and molecular phylogenetic analyses based on the DNA sequences of individual translation elongation factor 1‐alpha and β‐tubulin genes revealed that the recovered isolates produced macroconidia typical of four‐septate with a foot‐shaped basal cell and belonged to F. temperatum that is distinctly different from its most closely related species F. subglutinans and others within Gibberella fujikuroi complex species from maize. All the strains from this newly isolated species were able to infect maize and wheat in field, with higher pathogenicity on maize. Mycotoxin determination of maize grains infected by the strains under natural field condition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry analyses showed that among fifteen mycotoxins assayed, two mycotoxins fumonisin B₁ and B₂ ranging from 9.26 to 166.89 μg/g were detected, with massively more FB₂ mycotoxin (2.8‐ to 108.8‐fold) than FB₁. This mycotoxin production profile is different from that of the Belgian population in which only fumonisin B₁ was barely detected in one of eleven strains assayed. Comparative analyses of the F. temperatum and F. subglutinans strains showed that the highest fumonisin producers were present among the F. temperatum population, which were also the most pathogenic to maize. These results suggested a need for proper monitoring and controlling this species in the relevant maize‐growing regions.     

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.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Leaf Spot on Sweet Potato (Ipomoea batatas) Caused by Stemphylium solani,a New Disease in China

During 2010–2011, a severe leaf spot disease of sweet potato (Ipomoea batatas) was found in Haikou City, Hainan province of China. The disease is characterized with large, irregular, brown, necrotic lesions on the margin or in the centre of leaves. A species of Stemphylium was consistently recovered from pieces of symptomatic tissues on PDA. Based on morphological characteristics and molecular identification by rDNA‐ITS gene analysis, the fungal species was identified as Stemphylium solani Weber, and its pathogenicity was confirmed by Koch's postulates. This is the first report of leaf spot on sweet potato caused by S. solani in China.     

Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC‐NBS‐LRR resistance gene of Malus × robusta 5

The fire blight susceptible apple cultivar Malus × domestica Borkh. cv. ‘Gala’ was transformed with the candidate fire blight resistance gene FB_MR5 originating from the crab apple accession Malus × robusta 5 (Mr5). A total of five different transgenic lines were obtained. All transgenic lines were shown to be stably transformed and originate from different transgenic events. The transgenic lines express the FB_MR5 either driven by the constitutive CaMV 35S promoter and the ocs terminator or by its native promoter and terminator sequences. Phenotyping experiments were performed with Mr5‐virulent and Mr5‐avirulent strains of Erwinia amylovora, the causal agent of fire blight. Significantly less disease symptoms were detected on transgenic lines after inoculation with two different Mr5‐avirulent E. amylovora strains, while significantly more shoot necrosis was observed after inoculation with the Mr5‐virulent mutant strain ZYRKD3_1. The results of these experiments demonstrated the ability of a single gene isolated from the native gene pool of apple to protect a susceptible cultivar from fire blight. Furthermore, this gene is confirmed to be the resistance determinant of Mr5 as the transformed lines undergo the same gene‐for‐gene interaction in the host–pathogen relationship Mr5–E. amylovora.     

The rice/maize pathogen Cochliobolus spp. infect and reproduce on Arabidopsis revealing differences in defensive phytohormone function between monocots and dicots

The fungal genus Cochliobolus describes necrotrophic pathogens that give rise to significant losses on rice, wheat, and maize. Revealing plant mechanisms of non‐host resistance (NHR) against Cochliobolus will help to uncover strategies that can be exploited in engineered cereals. Therefore, we developed a heterogeneous pathosystem and studied the ability of Cochliobolus to infect dicotyledons. We report here that C. miyabeanus and C. heterostrophus infect Arabidopsis accessions and produce functional conidia, thereby demonstrating the ability to accept Brassica spp. as host plants. Some ecotypes exhibited a high susceptibility, whereas others hindered the necrotrophic disease progression of the Cochliobolus strains. Natural variation in NHR among the tested Arabidopsis accessions can advance the identification of genetic loci that prime the plant’s defence repertoire. We found that applied phytotoxin‐containing conidial fluid extracts of C. miyabeanus caused necrotic lesions on rice leaves but provoked only minor irritations on Arabidopsis. This result implies that C. miyabeanus phytotoxins are insufficiently adapted to promote dicot colonization, which corresponds to a retarded infection progression. Previous studies on rice demonstrated that ethylene (ET) promotes C. miyabeanus infection, whereas salicylic acid (SA) and jasmonic acid (JA) exert a minor function. However, in Arabidopsis, we revealed that the genetic disruption of the ET and JA signalling pathways compromises basal resistance against Cochliobolus, whereas SA biosynthesis mutants showed a reduced susceptibility. Our results refer to the synergistic action of ET/JA and indicate distinct defence systems between Arabidopsis and rice to confine Cochliobolus propagation. Moreover, this heterogeneous pathosystem may help to reveal mechanisms of NHR and associated defensive genes against Cochliobolus infection.     

Molecular characterization of Streptococcus agalactiae strains isolated from fishes in Malaysia

Aims

The aim of this study was to characterize Streptococcus agalactiae strains that were isolated from fishes in Malaysia using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (REP‐PCR) techniques.

Methods and Results

A total of 181 strains of Strep. agalactiae isolated from red hybrid tilapia (Oreochromis sp.) and golden pompano (Trachinotus blochii) were characterized using RAPD and REP‐PCR techniques. Both the fingerprinting techniques generated reproducible band patterns, differing in the number and molecular mass amplicons. The RAPD technique displayed greater discriminatory power by its production of more complex binding pattern and divided all the strains into 13 groups, compared to 9 by REP‐PCR technique. Both techniques showed the availability to differentiate the genetic profiles of the strains according to their geographical location of origin. Three strains of Strep. agalactiae that were recovered from golden pompano showed a genetic dissimilarity from the strains isolated from red hybrid tilapia, while the strain of ATCC 27956 that recovered from bovine displayed a unique profile for both methods.

Conclusions

Both techniques possess excellent discriminative capabilities and can be used as a rapid means of comparing Strep. agalactiae strains for future epidemiological investigation.

Significance and Impact of the Study

Framework as the guideline in traceability of this disease and in the search for potential local vaccine candidates for streptococcosis in this country.     

Investigating variability and behaviour of Colletotrichum gloeosporioides strains from lesions of coffee blister spot

Coffee blister spot has been associated with species from the Colletotrichum genus, but there is no information on the variability of isolates present on leaf lesions. This study evaluated a population of Colletotrichum gloeosporioides strains from blister spot lesions in Coffea arabica. Colletotrichum spp. isolates were collected from blister spot lesions on leaves of coffee trees from Catuaí and Topázio cultivars (Coffea arabica). Monosporic cultures were obtained from colonies with sporulation. A pathogenicity test was carried out by inoculation of pathogens on the leaves of young coffee plants. C. gloeosporioides strains were characterized by morphologial, cytological and physiological analyses. The molecular analysis was carried out using Inter‐Retrotransposon Amplified Polymorphism (IRAP) markers. C. gloeosporioides strains showed no pathogenicity on coffee plants and presented a wide variability in all traits evaluated. The presence of sexual strains, formation of CATs (conidial anastomosis tubes) among conidial strains and high mycelial compatibility among strains observed suggest the occurrence of sexual and asexual recombination. The role of these C. gloeosporioides strains on the lesions of coffee plant leaves is unclear.     

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.