Canker Symptoms Research Articles

Stem Canker on Cyclocarya paliurus Is Caused by Botryosphaeria dothidea.

Cyclocarya paliurus, an important endangered plant in China, has considerable medicinal, timber, and horticultural value. However, little is known about diseases that affect its health. In recent years, stem canker diseases on C. paliurus have been observed frequently in newly established nurseries in Jiangsu Province, China. Symptomatic trees showed elliptical, sunken lesions on the bark, with internal discoloration, leading to enlarging cankers with delineated margins. Pathogenicity tests with fungi isolated from symptomatic samples reproduced typical canker symptoms on both detached branches and potted plants of C. paliurus. Moreover, conidia from pycnidia of isolate ZB-23 could also cause stem canker on C. paliurus. Through combined morphological observation and DNA sequences of ITS region, β-tubulin, and translation elongation factor 1-α genes, the pathogen was identified as Botryosphaeria dothidea. Multigene maximum likelihood and maximum parsimony phylogenetic analyses further supported the identification of the pathogen. To our knowledge, this is the first report of B. dothidea causing stem canker on C. paliurus in China.

Quantitative PCR Assays Developed for Diaporthe helianthi and Diaporthe gulyae for Phomopsis Stem Canker Diagnosis and Germplasm Screening in Sunflower (Helianthus annuus).

Phomopsis stem canker of sunflower is caused by two fungal pathogens, Diaporthe helianthi and Diaporthe gulyae, in the United States. In this study, two quantitative PCR (qPCR) assays were developed to detect and quantify D. helianthi and D. gulyae in sunflower. The two assays differentiated the two fungi from each other, other species of the genus Diaporthe, and pathogens, and they have high efficiency (>90%). The qPCR assays detected the two pathogens on plant samples exhibiting Phomopsis stem canker symptoms sampled from commercial sunflower fields in Minnesota, Nebraska, North Dakota, and South Dakota. Furthermore, the assays were used to screen cultivated sunflower accessions for resistance to D. helianthi and D. gulyae. The disease severity index (DSI) of the accessions significantly correlated (P < 0.0001) with the amount of pathogen DNA from the qPCR assays. The qPCR assays identified PI664232 and PI561918 to be significantly less susceptible (P ≤ 0.05) to D. helianthi and D. gulyae, respectively, when compared with the susceptible check cultivar HA 288, and this was in agreement with the DSI. These results suggest that the qPCR assays for D. helianthi and D. gulyae can be used as a reliable tool to diagnose Phomopsis stem canker and screen sunflower germplasm for disease resistance.

Identification of Pseudomonas Isolates Associated With Bacterial Canker of Stone Fruit Trees in the Western Cape, South Africa.

Bacterial canker is a common bacterial disease of stone fruit trees. The causal agents responsible for the disease include several pathovars in Pseudomonas syringae sensu lato and newly described Pseudomonas species. Pseudomonad strains were isolated from symptomatic stone fruit trees, namely apricot, peach, and plum trees cultivated in spatially separated orchards in the Western Cape. A polyphasic approach was used to identify and characterize these strains. Using a multilocus sequence typing approach of four housekeeping loci, namely cts, gapA, gyrB, and rpoD, the pseudomonad strains were delineated into two phylogenetic groups within P. syringae sensu lato: P. syringae sensu stricto and Pseudomonas viridiflava. These results were further supported by LOPAT diagnostic assays and analysis of clades in the rep-PCR dendrogram. The pseudomonad strains were pathogenic on both apricot and plum seedlings, indicative of a lack of host specificity between Pseudomonas strains infecting Prunus spp. This is a first report of P. viridiflava isolated from plum trees showing symptoms of bacterial canker. P. viridiflava is considered to be an opportunistic pathogen that causes foliar diseases of vegetable crops, fruit trees, and aromatic herbs, and thus the isolation of pathogenic P. viridiflava from twigs of plum trees showing symptoms of bacterial canker suggests that this bacterial species is a potentially emerging stem canker pathogen of stone fruit trees in South Africa.

First Report of Eutypella Canker Caused by Eutypella parasitica on Acer campestre in Italy

In autumn 2016, typical Eutypella canker symptoms were first observed on ∼30-year-old field maple (Acer campestre L.) trees growing in a mixed ash-maple seminatural forest in northeastern Italy (Paciuch/Pacuh, Udine/Videm, 46°10′43″N, 13°38′13″E). In subsequent surveys carried out in 2017, the disease was observed in another 20 trees along a 36-km-long transect parallel to the Italian-Slovenian border (Drenchia/Dreka to Gorizia/Gorica, 46°10′20″N, 13°38′30″E to 45°57′18″N, 13°36′40″E). Symptomatic trees showed approximately 80-cm-long elongated cankers with the bark remaining firmly attached to the stem and with the presence of subcortical white mycelial fans and black perithecial necks. The morphological features of the teleomorph—perithecia 502 (365 to 638) µm, asci 87.9 (65.3 to 106.5) × 9.0 (7.3 to 11.5) µm, and ascospores 9.3 (7.8 to 11.2) × 3.2 (2.5 to 3.7) µm (n = 40)—matched the description of Eutypella parasitica R.W. Davidson & R.C. Lorenz reported by Davidson and Lorenz (1938). To isolate the pathogen, small fragments of mycelial fans and symptomatic inner bark and woody tissues (4 to 5 mm²) taken from the margin of 20 cankers were placed into Petri dishes (90 mm O) containing potato dextrose agar (PDA, 39 g/liter, Oxoid, Basingstoke, U.K.). All isolates developed white colonies with a dense mycelium appressed to the agar surface that became light gray after 20 to 25 days at 25°C in the dark. The hyaline, filiform, irregularly curved, and aseptate conidia were exuded in a yellow matrix and measured 37.8 (26.9 to 48.5) × 2.0 (1.4 to 2.6) μm (n = 50). The identity of the 20 fungal isolates obtained (one from each canker) was confirmed by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. Fungal DNA extraction, PCR amplification reactions, and DNA sequencing were carried out as reported by Linaldeddu et al. (2016). BLAST searches against GenBank showed 100% identity with reference sequences of E. parasitica, including that of the ex-type culture CBS 210.39 (GenBank accession no. NR145214). The ITS sequence of a representative strain of E. parasitica, PC3, was deposited in GenBank (accession no. MN227194). The strain PC3 was stored on PDA slants under oil in the culture collection of the Dipartimento Territorio e Sistemi Agro-Forestali, University di Padua (Italy). Testing the pathogenicity of the strain (PC3) was undertaken by inoculating ten 3-year-old field maple seedlings. Ten additional seedlings were inoculated with a Slovenian strain of E. parasitica (T1-1/3NO) obtained from Acer pseudoplatanus L. (GenBank accession no. MN607439). The part of the stem to be inoculated was surface disinfected with 90% ethanol, and a small piece (4 × 4 mm) of bark was removed with a flamed scalpel. An agar mycelium plug (3 × 3 mm) taken from the margin of an actively growing colony on PDA was placed on the wound, covered with cotton wool soaked in sterile water, and finally wrapped in a piece of aluminum foil. A sterile PDA plug was used instead of the mycelial inoculum in 10 control seedlings. Plants were kept under field conditions at 13 to 37°C and watered regularly for 2 months. At the end of the experiment, all plants inoculated with E. parasitica were symptomatic and showed open cankers and dark brown lesions in the xylem spreading up and down from the inoculation site. The average lesion size was 2.40 ± 0.71 cm for the Italian strain (PC3) and 2.70 ± 0.55 cm for the Slovenian strain (T1-1/3NO). Both strains were successfully recovered from all the inoculated plants, fulfilling Koch’s postulates. No disease symptoms were detected on control seedlings, and the wounds had begun to heal. E. parasitica, a pathogen probably native to North America, was detected for the first time in Europe (Slovenia) in 2005 (Jurc et al. 2006) and thereafter reported in Austria, the Czech Republic, Croatia, Germany, Hungary, and Poland (Cech et al. 2016; Cerný et al. 2017). This is the first record of E. parasitica in Italy, where maple tree species (mainly A. campestre, A. pseudoplatanus, and A. platanoides L.) are naturally present in the mountain forests in the Alps and intensively cultivated for timber production. The wide spread and virulence of this pathogen together with its recent detection in the same sites on A. pseudoplatanus represents a serious threat to maple ecosystems in the Alpine region.

Identification and characterization of bacterial strains associated with diseased oak trees in Northern Iran

AbstractOak decline syndrome is characterized by periodic occurrences of decline and death of oaks over widespread areas. An outbreak of a new emerging disease on oak trees was reported in the Hyrcanian forest of Iran (Mazandaran and Golestan provinces) that showed stem bleeding and canker symptoms. Bacterial isolates were characterized through biochemical and physiological tests, protein electrophoresis, DNA fingerprinting (rep‐PCR, ERIC and BOX primers) and sequencing of 16S rRNA and MLSA (multilocus sequencing analysis) for housekeeping genes (gyrB, infB and atpD). A complex community of the genus Brenneria spp. (Brenneria goodwinii, Brenneria roseae subsp. roseae, Brenneria sp. and Brenneria nigrifluens) and a few isolates in the genus Gibbsiella were identified as major groups involved. Isolate differentiation was more accurate using concatenated partial gene sequences within the main groups. All bacterial isolates showed hypersensitivity reactions (HR) on Pelargonium leaves (Pelargonium × hortorum). Pathogenicity studies of different Brenneria and Gibbsiella strains revealed that they have potential to cause the disease in oak seedlings and devastating oak canker and stem bleeding symptoms in northern Iran. Due to the presence of several potentially pathogenic agent(s) associated with the oak decline, identification of the principal agent(s) is of major interest. To our knowledge, this is the first report of potentially pathogenic bacteria associated with oak bleeding and canker in Iran.

Molecular Aspects of an Emerging Poplar Canker Caused by Lonsdalea populi.

The Gram-negative bacterium Lonsdalea populi causes a lethal disease known as bark canker on Populus × euramericana in China and Europe. Typical symptoms of bark canker include an abundant white-colored fluid, which oozes from the infected tissues. The availability of the genomic sequence of the bacterium provided the necessary resource to launch genome-scale investigations into the mechanisms fundamental to pathogenesis. Functional analyses of a diverse group of genes encoding virulence factors and components of signaling pathways indicate that successful bark infection depends on specific responses by the pathogen to various stresses, including oxidative stress. Although physiology of resistance is well studied, the molecular processes underlying the defense responses and the genetic basis of resistance to L. populi and in other poplar species remain largely unknown. Control of the disease has relied on chemical measures. Due to the genetic amenability of Lonsdalea and poplar, this pathosystem will become an important model system to unravel molecular mechanisms of bacterial pathogenicity on woody plants. Increased understanding of pathogenesis and signaling in the interaction will facilitate the management of this kind of poplar canker.

Botryosphaerialean fungi causing canker and dieback of tree hosts from Mount Yudu in China

The order Botryosphaeriales includes many latent fungal pathogens with a wide range of woody hosts. The taxonomy of these pathogens has been difficult due to the use of poorly informing markers in phylogenetic analyses and the lack of good morphological characters. Many genera and families in this order have not yet been systematically studied in different hosts and from different regions. In this study, a total of 29 fungal strains from the Aplosporellaceae and Botryosphaeriaceae were isolated from branches or twigs with symptoms of canker and dieback disease in Mount Yudu of China. Morphology and multigene analyses (ITS, LSU and TEF1-α) indicated five distinct lineages, including Aplosporella javeedii, Botryosphaeria dothidea, Diplodia quercicola sp. nov., Phaeobotryon aplospora sp. nov. and Phaeobotryon rhois. Diplodia quercicola is characterized by multiloculate conidiomata, producing oblong to cylindrical, thick-walled, hyaline, aseptate conidia. Phaeobotryon aplospora is characterized by pulvinate, multiloculate conidiomata, producing ellipsoid to oblong, brown, aseptate conidia. The new species differ from related species phylogenetically and ecologically and in morphological features.

Canker and Wood Rot Pathogens Present in Young Apple Trees and Propagation Material in the Western Cape of South Africa.

Canker and wood rot pathogens cause dieback and, in severe cases, the death of young apple trees. Recently, a higher occurrence of cankers was observed on 1-year-old apple trees in the Western Cape Province of South Africa. This study aimed to assess the phytosanitary status of nursery trees and propagation material as possible inoculum sources for canker pathogens. Thirteen 1-year-old apple orchards showing canker or dieback symptoms were sampled. Certified nursery apple trees were collected from four nurseries as well as scion and rootstock mother plant material. Isolations were made from the discoloration observed in the vascular tissue of the plant parts and from asymptomatic material. Possible canker and wood rot species were identified with PCR and sequence comparisons of the relevant gene regions and phylogenetic analyses. Similar canker and wood rot species were isolated from 1-year-old diseased apple trees, nursery apple trees, and the propagation material. Forty-five fungal species associated with canker or wood rot symptoms were identified. The top five most abundant fungal species found causing disease on commercial 1-year-old trees were also found in high numbers causing latent infection in certified apple nursery trees. These species were Didymosphaeria rubi-ulmifolii sensu lato, Diplodia seriata, Schizophyllum commune, Didymella pomorum, and Coniochaeta fasciculata, with D. rubi-ulmifolii sensu lato being the dominant species in both sampling materials. In all, 65% of certified nursery apple trees, 5% of scion shoots used for budding, and 21% of rooted rootstock cuttings from layer blocks had latent infections of canker and wood rot pathogens. Pathogenicity trials were conducted with isolates of 39 species, inoculated onto 2-year-old branches of 14-year-old Golden Delicious trees. All species caused lesions that were significantly longer than the control. This study confirmed the presence of canker and wood rot pathogens in apple propagation material as well as certified nursery apple trees, which will aid the improvement of management practices in nurseries.

Diaporthalean fungi associated with canker and dieback of trees from Mount Dongling in Beijing, China.

Diaporthales is a fungal order comprising important plant pathogens, saprobes and endophytes on a wide range of woody hosts. It is often difficult to differentiate the pathogens in this order, since both the morphology and disease symptoms are similar among the various species. In the current study, we obtained 15 representative diaporthalean isolates from six tree hosts belonging to plant families Betulaceae, Fagaceae, Juglandaceae, Rosaceae, and Ulmaceae from Mount Dongling in China. Six species were identified residing in four families of Diaporthales (Diaporthaceae, Erythrogloeaceae, Juglanconidaceae and Melanconidaceae). Based on morphological comparison and the phylogenetic analyses of partial ITS, LSU, cal, his3, rpb2, tef1-α and tub2 gene sequences, we identified five known species (Diaporthe betulina, D. eres, D. rostrata, Juglamconis oblonga and Melanconis stilbostoma) and one novel species (Dendrostoma donglinensis). These results represent the first study of diaporthalean fungi associated with canker and dieback symptoms from Mount Dongling in Beijing, China.

Lasiodiplodia pseudotheobromae causes stem canker of Chinese hackberry in China

Chinese hackberry (Celtis sinensis Pers.) is an adaptable species widely growing in southern China. The symptoms of canker on stems of seedlings were discovered mid-July 2017 in Shuyang, Jiangsu Province. The diseased portions of the stems were dark brown due to discoloured xylem. Some seedlings showed symptoms of wilting, leaf fall, twig dieback, and tissue discolouration. The outbreak period was concentrated in July and August, suggesting that the disease spread during summer months. Possible fungal causal agents were isolated from naturally infected canker tissue and discoloured xylem. The isolate from xylem tissue with a high frequency (> 50%) was named Ls7 type. Pathogenicity tests were carried out on 4-year-old seedlings. The symptoms of canker began to develop 20 days after inoculation with Ls7 isolate and by day 35, there were dark, enlarged longitudinal lesions. A phylogenetic tree of the isolate was developed using the internal transcribed spacer, elongation factor-1α (tef1-α), β-tubulin gene (TUB) and RNA polymerase II subunit primer genes (RPB2). Based on morphological features and phylogenetic information, the pathogen was identified as Lasiodiplodia pseudotheobromae. This is the first report of L. pseudotheobromae causing canker on Chinese hackberry stems in China.

First Report of Lasiodiplodia pseudotheobromae Causing Canker and Shoot Dieback on Apple in China

In July 2017, apple trees (Malus pumila Mill.) with canker and shoot dieback symptoms on side branches were observed in several young orchards in Yantai, Shandong Province, China. Symptoms included shoot dieback, discoloration of xylem tissues, cankers, and sunken necrotic bark lesions, which surrounded the branches and resulted in the death of the branches. The disease is symptomatically different from canker diseases of apple earlier described. Small pieces (4 to 5 mm) of necrotic tissues from the shoot cankers were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid. White colonies developed from symptomatic tissues but became dark gray mycelia with age. Pycnidia were produced on sterilized apple twigs on 2% water agar after incubation for 3 weeks at 25°C. Conidia were unicellular, ellipsoidal, initially hyaline, and became dark brown and developed a central septum with age. Conidia measured 21.3 to 31.9 × 12.4 to 16.9 µm (n = 50). On the basis of morphological characteristics, the isolates were identified as Lasiodiplodia sp., a member of the Botryosphaeriaceae family (Alves et al. 2008; Phillips et al. 2013). For further identification, total DNA was extracted from the mycelium of three monoconidial isolates (La1, La2, and La3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), and translation elongation factor 1-α gene (TEF1) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-688F/EF1-1251R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The sequences (GenBank accession nos. MK408640 to MK408642 for ITS, MK446722 to MK446724 for TUB, and MH829861 to MH829863 for TEF1) were aligned using BLASTn in GenBank, showing 99 to 100% homology with the sequences of ITS (EF622077), TUB (EU673111), and TEF1 (EF622057) from L. pseudotheobromae isolate CBS 116459 (Alves et al. 2008). The pathogenicity testing of three isolates was conducted using the wound inoculation method both on living apple trees and detached twigs. Thirty 2-year-old twigs were wounded with a scalpel on the phloem tissues and inoculated by placing 5-mm² agar plugs containing 2-day-old cultured hyphae or 10 μl of an inoculum suspension (10⁵ conidia/ml), which were then sealed with Parafilm. The same number of wounded twigs was inoculated with uncolonized PDA plugs or sterile water as controls. After 7 days, necrotic lesions (35 to 60 mm in diameter) developed around the inoculation points, which resembled the symptoms on infected apple shoots in the field. No symptoms were observed on the control twigs, and the pathogenicity test was conducted thrice. The pathogen was reisolated from symptomatic twigs and was reidentified as L. pseudotheobromae on morphological and molecular analysis. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), to our knowledge, this is the first report of the pathogen causing canker and shoot dieback on apple in China. This report is important for the new research aiming at management of apple canker disease.

Diaporthe eres causes stem cankers and death of young apple rootstocks in Canada

In the spring of 2018, necrotic and canker symptoms were observed on stems of young apple trees that were bench-grafted by growers on four rootstocks (EMLA 9, Supporter 4, M9-T337 and Pajam 2) in Annapolis Valley, Nova Scotia. The objective of this study was to isolate and identify the causal organism of the disease on apple rootstocks. The diseased tissue obtained from eight infected plants of the four rootstocks showing visible necrotic and canker symptoms was used for microorganism isolation on potato dextrose agar (PDA). More than 90% of the isolates on PDA yielded a similar type of fungal colony. Based on the colony and spore morphology and presence of both α and β spores, Diaporthe was proposed as a major pathogen from the symptomatic tissue. Analysis of partial sequences of four unlinked loci DNA-lyase (APN2), histone-3 (HIS), translation elongation factor 1 alpha (TEF1-α) and the internal transcribed spacer (ITS) 5.8S region of rDNA of the representative fungal isolates from young apple trees confirmed its identity as D. eres (Phomopsis fukushii). The pathogenicity of the representative D. eres isolate was confirmed on 1-year-old young apple trees bench-grafted on four different rootstocks in greenhouse tests. Necrotic and sunken canker symptoms like those observed on the nursery-grown young apple trees appeared on stems of all inoculated plants. The necrotic and canker lesions were observed 3 days after inoculation; they spread both directions from the inoculation point and led to the death of the young plants after 4 weeks. To our knowledge, this is the first report of D. eres causing stem canker and death of young apple rootstock in Canada.

Characterization of Fusarium and Neocosmospora Species Associated With Crown Rot and Stem Canker of Pistachio Rootstocks in California.

California produces 99.1% of pistachios grown in the United States, and diseases affecting pistachio rootstocks represent a constant challenge to the industry. Field surveys of fungi associated with pistachio rootstocks with symptoms of crown rot and stem canker in three central California counties followed by phylogenetic analyses of translation elongation factor 1-α and second largest subunit of RNA polymerase II gene fragments identified three Fusarium species (Fusarium equiseti, Fusarium oxysporum, and Fusarium proliferatum) and two Neocosmospora species (Neocosmospora falciformis and Neocosmospora solani). F. oxysporum and N. falciformis were the fungal species most frequently recovered from symptomatic pistachio trees. Inoculations of detached twigs of cultivar Kerman pistachio Pioneer Gold I and clonal University of California, Berkeley I (UCBI) rootstocks showed that all five species could colonize pistachio wood and cause vascular discolorations. Pathogenicity tests in potted pistachio trees completed Koch's postulates and confirmed that F. oxysporum, F. proliferatum, N. falciformis, and N. solani were capable of producing rot and discoloration in stems of clonal UCBI rootstocks, the most widely planted pistachio rootstock in California. To our knowledge, this study is the first to present insights into the biodiversity and biology of Fusarium and Neocosmospora species associated with pistachio trees in California.

Latent Infection of Valsa mali in the Seeds, Seedlings and Twigs of Crabapple and Apple Trees is a Potential Inoculum Source of Valsa Canker

A real-time quantitative PCR assay using a species-specific primer pair was developed to rapidly and accurately quantify Valsa mali, the causative pathogen of apple Valsa canker (AVC), in crabapple seeds, crabapple seedlings, apple twigs and apple seeds. Surveys were conducted in different regions, and crabapple or apple seeds were collected for V. mali detection by qPCR assay. Our results showed that 12.87% to 49.01% of crabapple seeds collected from different regions were positive for V. mali. The exopleura and endopleura were the two major areas of V. mali infection in crabapple seeds. The presence of V. mali infection in crabapple seeds was also confirmed by a high-throughput sequencing approach. With the growth of crabapple seedlings, the concentration of V. mali gDNA in crabapple seedlings gradually increased until eight or more leaf blades emerged. One-year-old twigs from an apple scion nursery were infected with V. mali, and only apple seeds from infected apple trees showing evident Valsa canker symptoms carried V. mali. In conclusion, this study reports that crabapple seeds and apple seeds carried V. mali as latent inoculum sources. V. mali infected not only apple tissues but also crabapple seedlings, which are the rootstocks of apple trees. This study indicated that the inoculum sources for AVC vary. Application of a novel qPCR assay can potentially improve the accuracy of early diagnosis, and is helpful to reveal the epidemic regularity of AVC.

Field Identification of Citrus Canker Symptoms and Decontamination Procedures

Citrus canker, caused by a bacterial pathogen Xanthomonas axonopodis pv. citri, is a serious disease of most citrus varieties. The disease causes necrotic lesions on leaves, stems, and fruit (Figure 1 and Figure 2). Severe infestation can cause defoliation, premature fruit drop, twig dieback, general tree decline, and very bad blemishes on fruit. Trees infected with citrus canker become weak, unproductive, and unprofitable. This document is PP-214, one of a series of the Plant Pathology Department, UF/IFAS Extension. Original publication date October 2005.&#x0D; PP-214/PP136: Field Identification of Citrus Canker Symptoms and Decontamination Procedures (ufl.edu)

Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β‐tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov.

First Report of Twig Dieback Caused by Neopestalotiopsis clavispora on Blueberry in Korea

Blueberry (Vaccinium corymbosum) is an economically important fruit with increasing cultivation in Korea, expanded to about 4,000 ha in 2017. In January 2018, dieback symptoms were observed on young twigs and stems of blueberry plants at an orchard located in Goheung, Jeonnam Province, Korea. Initial red spots developed into larger lesions with brown margins, which gradually enlarged and developed into gray cankers. Affected twigs showed blight symptoms that gradually expanded along the stems, culminating in dieback. No acervuli and pycnidia were observed on diseased twigs and/or stems. Ten twigs displaying typical blight lesions were collected from each of 10 individual V. corymbosum ‘Duke’ plants. Small pieces of tissue were cut from the edge of each lesion, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterilized water, and cultured on potato dextrose agar at 25°C. A total of 19 isolates were identified by morphological examination and molecular analysis. Colonies were white with cottony aerial mycelium, irregular margins, and light yellow on the reverse side of the plate, with black, globular acervuli (120 to 400 μm, average 230 μm) distributed in concentric circles. Pycnidial conidiomata were swollen and globose to oval. Conidia were straight to fusiform, measuring 19.5 to 30.1 × 5.0 to 11.0 μm (n = 50) and consisted of five cells with the three median cells being much darker than the end cells. The three median cells were doliform, rugose, and versicolored and had septa darker than the rest of the cell. The apical cell was hyaline, conic, and subcylindric, with two to three apical hyaline appendages (average 26.5 μm). The sexual stage was observed. These morphological characteristics are consistent with those of Neopestalotiopsis clavispora (Borrero et al. 2018). The ITS (MH423964-82), TUB2 (MH423945-63) and tef1 (MH423926-44) sequences matched with N. clavispora strains LB14 (99%; KY319134), FC-DB1 (99%; KY568918), and TOR-802-803-804 (99%; KU096881), respectively. Using MEGA X program (Kumar et al. 2018), maximum likelihood analysis based on the concatenated ITS, TUB2, and tef1 sequences placed the isolates within a clade comprising N. clavispora (G.F. Atk.) (Jayawardena et al. 2016; Song et al. 2014). To confirm pathogenicity of the isolates, three twigs of three 1-year-old V. corymbosum ‘Duke’ were inoculated by spraying conidial suspension (1 × 10⁵ conidia/ml) with and without wounding using a sterilized needle. Inoculated plants were maintained in a plastic bag for 16 h in a greenhouse with 16-h photoperiod, temperature between 18 and 30°C, and humidity maintained at 75%. Red-brown specks were observed on both the wounded and nonwounded twigs at 3 days postinoculation (dpi) and 2 months postinoculation, respectively. In the wound-inoculated twigs, the developed specks gradually grew larger at 7 to 14 dpi. After 14 dpi, the twigs showed gray canker symptoms typically observed on naturally infected plants. Control plants showed no symptoms on both the wounded and nonwounded plants. The pathogen was successfully reisolated using the methods described above and identified via analysis of the three genes and morphological characteristics. This pathogen has been previously reported to cause twig blight on blueberry in Chile, Uruguay, China, and Spain (Borrero et al. 2018; Chen et al. 2016; Espinoza et al. 2008; Gonzalez et al. 2012). To our knowledge, this is the first report of N. clavispora causing twig dieback on V. corymbosum ‘Duke’ in Korea, and this information will aid development of disease control strategies.

Characterization of a novel victorivirus isolated from the phytopathogenic fungus Botryosphaeria dothidea.

Botryosphaeria dothidea is an important pathogenic fungus that causes serious diseases in fruits and trunks of many wood plant species worldwide. In this study, 28 B. dothidea strains isolated from pear trunk samples showing stem wart or canker symptoms were used to detect double-stranded RNA (dsRNA) viruses. The dsRNA bands with the size of ~ 1.0 to ~ 6.0 kbp were examined from ten strains. Here, we reported a novel dsRNA mycovirus, tentatively named as Botryosphaeria dothidea victorivirus 2 (BdVV2), isolated from the B. dothidea strain MSD53. BdVV2 contained spherical virions that were ~ 38nm in diameter consisting of a single linear dsRNA genome of 5,090bp in length. The BdVV2 genome contained two overlapping open reading frames (ORFs) encoding a putative coat protein (CP) and an RNA-dependent RNA polymerase (RdRp), which shared significant amino acid identities of 68% and 60% with the corresponding proteins of Sphaeropsis sapinea RNA viruses 1 (SsRV1). Phylogenetic analyses based on the aa sequences of CP and RdRp both showed that BdVV2 was phylogenetically related to the members of the genus Victorivirus in the family Totiviridae. BdVV2 is thus a novel victorivirus isolated from the phytopathogenic fungus B. dothidea.

Etiology of leaf spot and fruit canker symptoms on stone fruits and nut trees in Iran

During July 2015 to June 2017, 63 walnut and 94 stone fruit orchards were surveyed in Iran to identify and characterize the causal agent of a disease inducing leaf spot and fruit canker symptoms. Altogether, 189 Prunus spp. and 141 walnut samples (i.e. leaves, branches and fruits) were collected from 10 Provinces in northern, northwestern, central and southern Iran. Forty-three yellow-pigmented Gram-negative bacterial isolates were recovered from symptomatic plants. Only seven bacterial strains isolated from walnut in Mazandaran and Golestan Provinces were identified as X. arboricola pv. juglandis based on pathogenicity assays, specific PCRs, and multilocus sequence analysis. Based on the phenotypic features and phylogenetic analysis of gyrB gene sequences, strains isolated from stone fruit trees were identified as multiple Pantoea species (i.e. P. agglomerans, P. ananatis, P. vagans and P. pleuroti). None of these strains caused symptoms on Prunus spp. plantlets tested under greenhouse conditions. Although the occurrence of bacterial blight of walnut in Iran was ascertained using molecular-phylogenetic analysis, no evidence was obtained that the observed disease was the same as the bacterial spot and canker of stone fruits caused by X. arboricola pv. pruni in the country.

Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China.

Dendrostoma was recently proposed in Erythrogloeaceae (Diaporthales, Sordariomycetes), with all known members recorded as being plant pathogenic on economically important tree hosts. During our collections of Dendrostoma species in China, mild to severe canker symptoms were observed on sweet chestnut (Castaneamollissima) and oak (Quercus spp.) trees. Dead and dying plant tissues exhibiting Dendrostoma canker symptoms were sampled for fungal isolation. A total of 37 Dendrostoma isolates were obtained and analysed using morphological characteristics and molecular data (ITS, LSU, RPB2, TEF1-α). Based on these data, 10 novel clades could be distinguished, which also proved to represent morphologically distinct species described here as Dendrostomaaurorae, D.castaneae, D.castaneicola, D.chinense, D.dispersum, D. parasiticum, D.qinlingense, D.quercus, D.shaanxiense and D.shandongense spp. nov. A key to species of the genus is also provided.