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Tetrastigma hemsleyanum is a rare medicinal plant in China. Because it requires very strict environmental conditions to grow, it usually takes 3–5 years to form calabash-shaped roots and is difficult to meet the demand of Chinese medical market. Although it is known that endophytes can enhance the growth of the host plants, the molecular mechanism of the interplay between endophytes and their host plants growth is not completely understood yet. The aim of this study was to determine if endophytes can reduce the growth cycle and promote the medicinal ingredients in Tetrastigma hemsleyanum, and elucidate its underlying molecular mechanism. Endophytic fungi were isolated and identified from the calabash-shaped root of Tetrastigma hemsleyanum. The fermentation broth of 6 endophyte strains (named as TH09, TH12, TH14, TH15, TH17 and TH26) were added to the MS medium to culture axenic plantlets. After 30 days of culture, the net growth, expansin gene expression and the flavonoid content were measured. From the calabash-shaped roots, we isolated 31 endophytic fungi belonging to 10 genera: Fusarium, Rhizopycnis, Colletotrichum, Cylindrocarpon, Emericellopsi, Penicillium, Plectosphaerella, Aspergillus tubingensis, Alternaria and Sarocladium, of which Fusarium is the dominant genus. Addition of the fermentation broth from strains TH12, TH15 and TH26 significantly increased the plant growth. Strains TH15 and TH26’s fermentation broth increased the expression of Th-exp (an expansin gene), while addition of the fermentation broth from TH14 increased Th-exp expression in leaves and stems, but decreased Th-exp expression in the root. Flavonoid content was increased in the presence of fermentation broth from strains TH09, TH14, TH17 and TH26. Particularly, the fermentation broth from TH26 can promote plant growth, enhance Th-exp expression and increase the flavonoid content in Tetrastigma hemsleyanum. The results demonstrated that endophytic fungi from the calabash-shaped root of Tetrastigma hemsleyanum can regulate plant growth, expression of expansin gene and flavonoid content.

Crown and collar rot of apple trees is a destructive and widespread disease in most areas of the world. Surveys have been done to describe disease symptoms, estimate the disease incidence, and identify the pathogens associated with this disease in southern Syria. Disease incidence was 0.08 – 10 % in most studied sites, only in Alroom location it was up to 14.7 % in 2014 and 17.8 % in 2015, with average of 11.8 %. Symptoms included small pale green leaves, sparse foliage, and a reddish-brown discoloration of inner bark of the infected area at the base of infected tree. The isolated fungi belonged to genera and form genera Phytophthora, Rosellinia, Rhizoctonia, Phialophora, Acremonium, Pestalotiopsis, Cylindrocarpon and Verticillium. Phytophthora was isolated from all infected trees, and was the most frequent pathogen (53.7 % of total isolates). Phytophthora isolates recovered from crown cankers of apple were identified as P. cactorum (91.5 %) and P. cambivora (8.5 %). The results of this study are the first report of crown and collar root rot of apple in Syria.

We assessed whether the presence and abundance of plant pathogens and antagonists change in soil fungal communities along a land abandonment gradient. The study was carried out in the Cilento area (Southern Italy) at a site with three different habitats found along a land abandonment gradient: agricultural land, Mediterranean shrubland and woodland. For all microbiological substrates the colony forming units were about 3.1 × 106 g−1 soil for agricultural land and about 1.1 × 106 g−1 soil for Mediterranean shrubland and woodland. We found the following genera in all habitats: Cladosporium, Mortierella, Penicillium and Trichoderma. In agricultural land, the significantly most abundant fungus genera were Aspergillus, Fusarium, Cylindrocarpon and Nectria; in Mediterranean shrubland, Rhizopus and Trichoderma; and in woodland, Bionectria, Mortierella, Cladosporium, Diplodia, Paecilomyces, Penicillium and Trichoderma. We found a total of 8, 8 and 9 species of fungal antagonist, and 16, 6 and 6 species of fungal plant pathogens in agricultural land, Mediterranean shrubland and woodland respectively. Fungal plant pathogens decreased significantly over a land abandonment gradient, while we no found significant differences among fungal antagonists in the three habitats. We conclude that a decrease in the number of fungal pathogen species occurs when formerly cultivated areas are abandoned. On the other hand, fungal antagonists seem not to be affected by this process.

[EN] During a survey of black foot disease in Algerian grapevine nurseries, a collection of 79 Cylindrocarpon-like isolates were obtained. Based on morphology and DNA sequence data of histone H3 (his3), three species of Dactylonectria were identified including Dactylonectria torresensis (40 isolates), D. macrodidyma (24 isolates) and D. novozelandica (14 isolates). In addition, one isolate belonging to the genus Pleiocarpon was found and it is described here as a new species, Pleiocarpon algeriense, based on morphological features and DNA sequence data of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), large subunit nrDNA (LSU) and histone H3 (his3). This is the first time that these species are reported in Algeria. Pathogenicity tests, were conducted with representative isolates from each species. All of them were able to induce typical necrosis symptoms on grapevine cuttings. These results emphasize the urgent need to implement an integrated management strategy for black foot disease in Algerian grapevine nurseries in order to reduce the incidence of this disease on grapevine planting material and to prevent that it spreads to new grapevine production areas., Much of this work was supported by the laboratory of the Grupo de Investigacion en Hongos Fitopatogenos, Instituto Agroforestal Mediterraneo (IAM), Universitat Politecnica de Valencia (UPV), Spain. W. Aigoun-Mouhous thanks the University of Blida for funding the research stay in Valencia, Spain. G. Elena was supported by the Spanish post-doctoral grant Juan de la Cierva-Formacion. A. Cabral was supported by Portuguese national funds through FundacAo para a Ciencia e a Tecnologia grant SFRH/BPD/84508/2012 and FCT Unit funding UID/AGR/04129/2013. This work was also supported by EFRR "Multidisciplinary research to increase application potential of nanomaterials in agricultural practice" (No. CZ.02.1.01/0.0/0.0/16_025/0007314).

Resumo – O declínio de plantas jovens, causado pela ação conjunta da pérola-da-terra ou margarodes (Eurhizococcus brasiliensis) e fungos patogênicos (Cylindrocarpon, Phaeoacremonium e outros), é um problema grave em vinhedos do Sul do Brasil. Para diminuir as perdas provocadas por esse problema fitossanitário pode-se adotar o uso de porta-enxertos resistentes, mas seu uso não está difundido nessa região. Neste trabalho avaliou-se as características agronômicas do cultivar Moscato Embrapa enxertado nesses porta-enxertos em comparação com Paulsen 1103 e R99, utilizados como referência. De uma forma geral todos os porta-enxertos testados podem ser utilizados para produção de uva. Os porta-enxertos tropicais IAC 313 Tropical, IAC571-6 Jundiaí e IAC 766 Campinas induziram a brotação precoce comparada a Paulsen 1103, ao contrário de VR043-43 e Dog Ridge. Mesmo os porta-enxertos tropicais, sem dormência no inverno, comportam-se como porta-enxertos normais quando enxertados. O IAC 572 Jales tem a vantagem de não induzir a brotação precoce, mas provoca vigor excessivo da planta, exigindo práticas especiais de manejo para controlar o desenvolvimento vegetativo.

Black-foot disease, caused by Cylindrocarpon obtusisporum (Cook & Harkn.) Wollenweb., impacts young table and wine grape (Vitis vinifera) plantings throughout California. Two- to 5-year-old grapevines showed reduced vigor with small-sized trunks, shortened internodes, uneven wood maturity, sparse foliage, and small leaves with interveinal chlorosis and necrosis. In cross-section, trunks showed dark brown to black streaking in a few to most of the vascular elements. Symptoms included a reduction in root biomass and root hairs with sunken, necrotic root lesions. Pith of affected vines was compacted and discolored. Isolations made from roots, vascular elements, and pith tissue consistently yielded colonies of C. obtusisporum as verified by descriptions in standard texts. Koch's postulates were completed by dipping the roots of cv. Carignane seedlings in a 108 spore per ml suspension for 30 min. Plants were repotted in an artificial soil mix and held in a controlled environment facility at 24°C. Typical black-foot symptoms developed on 92% of the plants within 8 weeks. Control plants dipped in distilled water remained healthy. Cylindrocarpon destructans, a species closely related to C. obtusisporum, was first reported to cause “black-foot disease” on young vines in 1961 (2). In 1975, C. obtusisporum was reported to produce similar “black-foot” symptoms (1). We propose the common name Cylindrocarpon black-foot disease be used with both species. References: (1) S. Grasso et al. Vitis 14:36, 1975. (2) D. R. Maluta and P. Larignon. Viticulture 11:71, 1991.

In this work, 82 Cylindrocarpon isolates associated with black foot disease of grapevines in Spain were studied by means of phenotypical characterization, DNA analyses, and pathogenicity tests. Partial sequences of the beta-tubulin (BT) gene, BT1, were amplified using primers BT1a and BT1b. A unique and conserved 52-bp insertion in the BT1 sequence, which is a specific marker for C. macrodidymum, was found in 56 of the isolates. The rest of the isolates (26) were identified as C. liriodendri. The BT phylogeny grouped all the isolates of each species into two well-supported clades. Phenotypical data were subjected to multivariate factorial analysis. According to this study, the isolates were clearly separated into two groups which were in agreement with BT species identification. C. macrodidymum isolates were differentiated from C. liriodendri by producing fewer conidia, presenting longer and wider macroconidia, and lower growth rate at 5 and 10°C. Selected isolates of each species inoculated onto rooted cuttings of grapevine rootstock cv. 110 R caused typical black foot disease symptoms. This is the first report of C. liriodendri and C. macrodidymum causing black foot disease of grapevines in Spain.

Black foot disease, caused by Cylindrocarpon macrodidymum Halleen, Schroers & Crous, is reported damaging table and wine grapes (Vitis vinifera L.) for the first time in Chile. During the summer of 2006, 2- to 5-year-old grapevines showed reduced vigor, shortened internodes, and drying and dying shoots along with abnormal development of roots with growth parallel to the soil surface, necrotic root crowns, and development of secondary roots. Internal necrosis extended from the bark to the pith in diseased parts of the plants. Other symptoms included black discoloration of the wood, gum inclusions in xylem vessels, black streaks in the vascular tissue, and reduction in root biomass, with sunken, necrotic root lesions. Eighteen Cylindrocarpon isolates were collected from roots, vascular elements, and pith tissue of grapevines cultivars (Flame Seedless, Red Globe, Thompson Seedless, Merlot, Carmenere, and Cabernet Sauvignon) from 12 locations in Chile. The isolates were identified on the basis of morphological features. All isolates produced micro- and macroconidia (one to three septa) and chlamydospores in short and intercalary chains (1,4), and by internal transcribed spacer (ITS1-5,8S-ITS4) rDNA and β -tubulin (BT1, and BT2) partial sequences, identical to those of C. macrodidymum (isolate USS074, GenBank Accession No. AY 997558 and isolate USSO150, GenBank Accession No. AY 997598) (2). Phylogenetic analyses placed these isolates in a clade closely related, but clearly distinct from other clades, to C. destructans and C. liriodendri (2,3). Pathogenicity tests were completed by drench inoculation onto 50 6-month-old rooted cuttings of ‘Red Globe’ with 25 ml of conidia suspension (106 conidia ml-1) obtained from four isolates. Ten control cuttings of ‘Red Globe’ were inoculated with an equal volume of sterile distilled water. The plants were incubated for 4 months in a controlled environment facility at 24°C. All isolates tested were pathogenic. In addition, they caused significant root rot (t-test of disease incidence, P = 0.0048) and no significant level of variation was detected between different isolates. C. macrodidymum was reisolated from the region of brown streaking in all the inoculated cuttings and was not isolated from the water-treated controls. To our knowledge, this is the first report of C. macrodidymum causing black foot disease on grapevine in Chile. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1966. (2) F. Halleen et al. Stud. Mycol. 50:431, 2004. (3) F. R. Mantiri et al. Can. J. Bot. 79:334, 2001. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.

Cylindrocarpon liriodendri and C. macrodidymum are the causal agents of grapevine black foot disease. Recently, a third species, C. pauciseptatum, has been isolated from roots of grapevine showing decline symptoms. Currently, reliable identification of isolates of these species through phenotypical characteristics has not been possible. The polymerase chain reaction (PCR)-based method developed in this study allows a quick and easy detection of Cylindrocarpon spp. associated with grapevine. Three primer pairs annealing to variable, partly species-specific sites of the internal transcribed spacer regions amplified species-specific PCR fragments of different sizes in C. liriodendri, C. macrodidymum, and C. pauciseptatum in a multiplex assay with DNA obtained with both quick and traditional extraction methods. They did not generate any PCR product in other fungal trunk pathogens or contaminants commonly associated with grapevines. When universal fungal ITS primers were used in a nested multiplex PCR, the three primer pairs also detected C. liriodendri, C. macrodidymum, and C. pauciseptatum in total DNA extracted from roots of inoculated grapevines. The designed methods can be used for the diagnosis of these fungi from pure culture or infected grapevines.

[EN] Weeds were sampled in grapevine rootstock mother fields, open-root field nurseries, and commercial vineyards of Albacete, Alicante, Castellón, Murcia, and Valencia provinces in Spain between June 2009 and June 2010 and evaluated as potential hosts of black-foot and Petri disease pathogens. Isolations were conducted in the root system and internal xylem tissues for black-foot and Petri disease pathogens, respectively. Cylindrocarpon macrodidymum was successfully isolated from the roots of 15 of 19 weed families evaluated and 26 of 52 weed species. Regarding Petri disease pathogens, one isolate of Phaeomoniella chlamydospora was obtained from Convolvulus arvensis, and three isolates of Cadophora luteo-olivacea were obtained from Bidens subalternans, Plantago coronopus, and Sonchus oleraceus. Pathogenicity tests showed that Cylindrocarpon macrodidymum isolates obtained from weeds were able to induce typical black-foot disease symptoms. When inoculated in grapevines, isolates of Cadophora luteo-olivacea and Phaeomoniella chlamydospora were also shown to be pathogenic on grapevine cuttings. Our ability to recover grapevine pathogens from vineyard weeds and to demonstrate pathogenicity of recovered strains on grape suggests that these weeds may serve as a source of inoculum for infection of grapevine. © 2011 The American Phytopathological Society., This research was financially supported by the projects RTA2007-00023-C04-03 and RTA2010-00009-C03-03 (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, INIA, Spain) and the European Regional Development Fund (ERDF). We thank A. Rustignoli and V. Garrigues for technical assistance and R. Raposo for providing valuable advice on the writing of this manuscript.

In November 2010, four grapevine plants of cv. Crimson from a vineyard located in Sevilla (south Spain) revealed trunk cankers. Several pathogens were isolated, including Cylindrocarpon liriodendri (2), Phaeoacremonium aleophilum (2), Pleurostomophora richardsiae, Neofusicoccum parvum, and Botryosphaeria dothidea (2). Among Botryosphaeriaceae fungi isolated on potato dextrose agar (PDA) were two types that did not fit the above mentioned species. Isolates of type 1 produced an abundant, gray mycelium with a diurnal zonation that gradually became dark olivaceous. Mycelium growth occurred from 5 to 37°C with an optimum at 28°C. Conidia were hyaline, fusiform, aseptate, thin walled, but gradually became obscured and septate with age, and measured (18.4-) 21.4 (-24.3) × (4.2-) 5.5 (-7.2) μm with a length/width (L/W) ratio of 4.0 ± 0.5 (n = 100). Isolates of type 1 were identified as N. mediterraneum (3). Single-spore cultures of type 2 developed a whitish, dense, aerial mycelium and remained white up to 10 days on PDA and darkened to gray thereafter. Mycelium growth occurred from 3 to 37°C with an optimum at 29 to 30°C. Conidia were hyaline, aseptate, thick walled, oblong to cylindrical, sometimes becoming light brown and one or two septate after discharge, and measured (24.6-) 30.2 (-42.8) × (10.9-) 14.3 (-18.6) μm with a L/W ratio of 2.1 ± 0.2 (n =100). Isolates of type 2 were identified as Diplodia corticola (1). Nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region and the -tubulin genes were used to confirm the identifications through BLAST searches in GenBank. Comparison of the sequences of types 1 and 2 showed 99 to 100% homology with N. mediterraneum (HM443604 (4) and GU251836) and D. corticola (AY268421 (1) and EU673117), respectively. Representative sequences of N. mediterraneum (JF949757 and JF949756) and D. corticola (JF949758 and JF949759) were deposited in GenBank. The pathogenicity of one representative isolate of each of N. mediterraneum and D. corticola was confirmed by inoculating 10 detached grapevine canes (averaging 12 mm in diameter and 30 cm long) per isolate. A shallow wound was made with a scalpel on the internodes. A colonized 6-mm agar plug, from the margin of an actively growing colony, was inserted in every wound and sealed with Parafilm. Ten grapevine canes controls received only sterile PDA agar plugs. Canes were maintained at 25°C and 70% humidity. After 5 weeks, all inoculated canes developed cankers and pycnidia around the inoculation site. Vascular necroses that developed on the inoculated canes were an average of 28.6 mm for N. mediterraneum and 27.7 mm for D. corticola. One-way analysis of variance and Tukey's test confirmed significant differences in the extent of vascular necroses. The average necroses length in the inoculated canes was significantly greater (P < 0.05) than the average length of discoloration induced by the simulated inoculation process in the control. Both pathogens were reisolated from all inoculated plants but not from controls. To our knowledge, this is the first report of N. mediterraneum and D. corticola as pathogens on grapevine in Spain. References: (1) A. Alves et al. Mycologia 96:603, 2004. (2) A. Aroca and D. Gramaje et al. Eur. J. Plant. Pathol. 126:165, 2010. (3) P. W. Crous et al. Fungal Planet. No. 19, 2007. (4) F. P. Trouillas et al. Plant. Dis. 94:1267, 2010.

Isolates morphologically identified as Cylindrocladiella parva were isolated from characteristic black foot symptoms on a grapevine (Vitis vinifera) rooted on 101-14 rootstock from Central Otago in 2005 and 101-14 rootstocks from a nursery in the Auckland Region in 2007 and 2008. On potato dextrose agar, the isolates initially produced cottony, white mycelia that turned grayish cream or golden cream within 10 days, the initially tawny colony undersides becoming dark brown with age. Conidia (0 to 1 septate; 16.4 to 17.0 [16.7] × 2.3 to 2.6 [2.5] μm) and abundant chlamydospores were produced. To confirm identity of the isolates, genomic DNA was extracted and the ribosomal DNA (rDNA) and β-tubulin gene were amplified and sequenced (3,4). Sequences of the PCR products were compared with sequences in GenBank. The rDNA (535 bp) and β-tubulin (297 bp) sequences of the four isolates were 100 and 99% identical, respectively, to reported sequences of C. parva in GenBank (AY793454, grapevine isolate (4)/AY793455 for rDNA; AY793486/AY793488, grapevine isolate (4)/AY793489/HM034822 for β-tubulin). Although C. parva was previously isolated from grapevines in New Zealand (2) and rootstocks of mature grapevines, cuttings, and graft unions of grafted young grapevines in South Africa (4), its role as a pathogen of Vitis spp. has not been confirmed (2,4). However, it has been reported as a pathogen of Eucalyptus spp. (1) and was also isolated from Telopea speciosissima and Macadamia integrifolia in New Zealand (2,4). The C. parva isolates were tested as a mixed inoculum (four isolates) for pathogenicity on roots of 10 grapevine rootstock plants each of cvs. 101-14 and Schwarzmann (Sch). The rootstocks were grown in potting mix for 4 months, after which the root systems of all vines were wounded with an asparagus knife with a sharp, square tip, driven vertically down into the soil at four equidistant locations approximately 8 cm from the trunk. Each plant was inoculated with 50 ml of the mixed-isolate conidial suspension (106/ml), or 50 ml water (controls), followed by 50 ml of water. After 7 months of growth, the plants were harvested. For C. parva-inoculated plants, internal blackening of the stem base tissue was observed. Isolations from surface-sterilized trunk bases recovered C. parva from four and nine plants of 101-14 and Sch, respectively, with C. parva infections in 25 and 48%, respectively, of the four wood pieces taken per plant. Plants inoculated with water had no blackening and no C. parva was isolated from their stem bases. Mean shoot dry weights of inoculated plants (17.9 and 15.0 g for 101-14 and Sch, respectively) were significantly lower (P = 0.035) than noninoculated controls (26.5 and 20.0 g for 101-14 and Sch, respectively). Mean root dry weights were reduced by C. parva inoculation, although not significantly (32.7 and 27.0 g for C. parva inoculated 101-14 and Sch, respectively, and 36.2 and 27.4 g for control 101-14 and Sch, respectively). To our knowledge, this is the first report of C. parva as a pathogen of grapevines (2,4) and suggests that along with Cylindrocarpon spp., C. parva is part of the pathogen complex responsible for black foot of grapevines. References: (1) P. W. Crous et al. Plant Pathol. 42:302, 1993. (2) P. D. Gadgil et al. Fungi on Trees and Shrubs in New Zealand. Fungal Diversity Press, Hong Kong, 2005. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) G. J. van Coller et al. Australas. Plant Pathol. 34:489, 2005.

During a survey for the sanitary status of stone fruits in southern Italy (Apulia region), symptoms of low vigor, sparse foliage, and chlorosis of leaves, frequently leading to decline or death of the plants, were observed on 3- to 5-year-old peach trees (Prunus persica) cvs. Tardi Belle, Zee Lady, and O'Henry grafted on GF677. Brown-to-black discolorations of the wood were observed in cross-sections of the trunks just below the graft union. Samples were collected from May to June 2010 from two symptomatic orchards in Brindisi and Foggia provinces. Small pieces of brownish, vascular wood and necrotic root tissues were surface disinfested, placed onto potato dextrose agar (PDA), and incubated for 7 days at 25°C in the dark. Single-conidial isolates were subsequently grown on PDA at 25°C for 10 days. Fungal colonies were presumptively identified as members of the genus Cylindrocarpon on the basis of their morphological and conidial characteristics. On PDA, the isolates developed abundant mycelium, which gradually became yellowish or partially brownish. Macroconidia were predominantly three septate, straight and cylindrical with both ends broadly rounded. Chlamydospores and ovoidal microconidia were observed on synthetic nutrient-poor agar (1). Sequence of the ribosomal internal transcribed spacer (ITS) region was obtained using universal primers (ITS6-ITS4) and deposited in GenBank (Accession No. HE577846). This sequence revealed 100% genetic identity with a sequence from Cylindrocarpon pauciseptatum Schroers & Crous (Accession No. EF607090), a recently described species (3). In nature, several species of the genus Cylindrocarpon affect a large number of woody plants, mainly grapevine, olive, and stone fruits, in which they attack the root surface (2). To verify Koch's postulates, the roots of 20 3-month-old peach seedlings (GF305) were dipped for 30 min in a spore suspension of the fungus (1 × 108 conidia ml–1). Seedlings were then transplanted in an artificial soil mix and held under controlled conditions in a greenhouse at 24°C. Typical black-foot symptoms developed on 92% of the inoculated plants within 3 months, whereas the control plants, whose roots had been dipped in distilled water, remained healthy. C. pauciseptatum was reisolated from infected tissues and internal vascular lesions of 45% of the inoculated plants, but none of the plants used as controls, fulfilling Koch's postulates. To our knowledge, this is the first report of this pathogen on peach in the Apulia Region of Italy. Currently, C. pauciseptatum is limited to a few orchards where presumably it was introduced with infected propagating material from extra-regional nurseries. C. pauciseptatum has the potential to negatively affect the stone fruit industry in Italy including reducing nursery production and productivity and vigor of trees in orchards, or even rapid death of young trees. References: (1) W. Gams et al. CBS Course of Mycology. 4th ed. Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, 1998. (2) M. E. S. Hernandez et al. Eur. J. Plant Pathol. 104:347, 1998. (3) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

The principal objective of this study was to determine the etiology of a canker disease in dormant stone fruit and apple tree seedlings maintained in refrigerated storage that has significantly impacted California fruit and nut tree nurseries. Signs and symptoms of the disease develop during storage or soon after planting, with subsequent decline and death of young trees. Isolations from both diseased and healthy almond and apple trees and Koch's postulates using stem segments of desiccation-stressed almond trees as hosts implicated Fusarium avenaceum and F. acuminatum as the primary causal agents. F. solani, Ilyonectria robusta, and Cylindrocarpon obtusiusculum were also capable of causing similar symptoms but were less frequently encountered in isolations of diseased tissue. Loss of bark turgidity in excised almond stem segments, as can occur in cold-stored seedlings, correlated with increased susceptibility to F. acuminatum, with maximum canker development occurring after relative bark turgidity dropped below a threshold of approximately 86%. Healthy almond trees, almond scion budwood, and a wheat cover crop used in fields where tree seedlings were grown and maintained until cold storage all possessed asymptomatic infections of F. acuminatum, F. avenaceum, and C. obtusiusculum as determined by activation following overnight freezing, cold storage, or desiccation.

Strawberry (Fragaria × ananassa Duchesne) is an economically important fruit crop in North Carolina for domestic consumption and export. In April 2012, outbreaks of a destructive root disease were observed in strawberry cv. Chandler in Buncombe, New Hanover, and Roman counties, North Carolina. Samples from Rowan (ID 13175) and Buncombe (ID 13193) counties submitted to the Plant Disease and Insect Clinic of the Department of Plant Pathology, North Carolina State University, exhibited yellowing and wilting of leaves and extensive root necrosis, and disease severity based on field symptoms ranged from 20 to 30%. To identify the pathogen, five small pieces of necrotic crown and root tissues were taken from each sample, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and plated onto potato dextrose agar (PDA) with 0.5 g liter–1 of streptomycin sulfate. Colonies developing from the tissue samples were transferred to PDA. Colonies from both samples were identical, grew relatively slowly, and gradually turned yellowish to partially brownish. After about 7 days, abundant conidia were formed. These were hyaline, mostly straight with both ends rounded, predominantly three septate, and 40 to 50 × 5 to 10 μm. Based on morphological characteristics, these isolates were identified as a species of Cylindrocarpon (1) To confirm the original identification of the fungus as a species of Cylindrocarpon, genomic DNA of both isolates was extracted from mycelia using DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and analyzed using PCR (2). The internal transcribed spacers (ITS)1 and (ITS)2 flanking the 5.8S rRNA regions were amplified and sequenced using universal primers ITS1 (forward) and ITS4 (reverse). The sequences of the 421 bp (GenBank KC847090 and KC847091) of both isolates were identical. Furthermore, a BLASTn search of these sequences showed homology of 99% with the sequences of Cylidrocarpon species (AB369421.1, AM419069.1, AM419074.1, AY295332.1, JN031017.1, JN253505.1, and JQ886422.1), To fulfill Koch's postulates, inoculum of each isolate was prepared and adjusted to 1.5 × 107 conidia/ml using a hemacytometer. ‘Chandler’ strawberry plants were grown in 25-cm diameter plastic pots (one seedling per pot) in the greenhouse and five 6-week-old plants were injected with conidia of each isolate into the base of crown using a 5-ml syringe. The plants were covered with clear plastic for 24 h and left on the greenhouse bench with a 16-h photoperiod and 25/20°C day/night temperatures and assessed for disease development 14 days after inoculation. The inoculated plants exhibited wilting and root necrosis, consistent with the symptoms observed on strawberry plants in the field. Control plants treated with distilled water remained healthy. Isolations were made from the inoculated plants and the fungus used for inoculation was recovered from all plants. The morphology of these isolates was in agreement with published descriptions of Cylindrocarpon (1). To our knowledge, this is the first report of a Cylindrocarpon sp. causing crown and root rot on strawberry in North Carolina and effective disease management strategies need to be explored. References: (1) C. D. Booth. Mycol. Pap. (CMI) 104:1, 1996. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Since 1999, the decline of American grapevines (Vitis labrusca L.) has been common in Rio Grande do Sul, Brazil (1). In August 2012, V. labrusca with black foot symptoms were collected in vineyards in the Serra Gaúcha Region. Symptomatic plants had low vigor, vascular lesions, delayed budding, and decline and death of vines. Symptomatic roots had necrotic lesions and reduced biomass. Fungal isolations were made from necrotic root and crown fragments (own-rooted cultivar) on potato dextrose agar (PDA) medium amended with 0.5 g L–1 streptomycin sulfate. Putative colonies of “Cylindrocarpon” pauciseptatum Schroers & Crous were obtained from single macroconidia isolations. Two isolates were used to confirm the identity of isolated colonies: Cy12UFSM and Cy13UFSM. After incubation in the dark for 10 days at 20°C, the isolated mycelial colonies, which were cottony white to felty in texture, became dark orange to brown. Both isolates produced chlamydospores in chains at 40 days. Chlamydospores of Cy12UFSM and Cy13UFSM were 9 to 12 μm and 5 to 11.5 μm in diameter. Sporodochia formation on carnation leaf agar (CLA) medium was observed after 30 days. To encourage development of conidia, the isolates were grown on spezieller nährstoffarmer agar (SNA) medium for five weeks at 20°C with addition of two pieces of 1 cm2 filter paper. Microconidia of Cy12UFSM were 4 to 8.5 × 3.5 to 5 μm and those of Cy13UFSM were 3.5 to 7.5 × 3 to 5 μm. Macroconida were predominantly 3-septate (Cy12UFSM was 36 to 45 × 7.5 to 9 μm and Cy13UFSM was 30 to 38 × 7.5 to 8 μm), but 1-, 2- septate macroconidia were observed. The sizes of the three spore types and colony morphology for our isolates were similar to those described by Schroers et al. (3) for “C.” pauciseptatum. To further confirm the identity of Cy12UFSM and Cy13UFSM, multi-gene DNA sequence analysis (rDNA-ITS, β-tubulin, and histone H3) was conducted using primer pairs ITS1 and ITS4 (4), Bt2a and Bt2b, and H3-1a and H3-1b (2), which amplify the ITS1-5.8S rRNA-ITS2 genes, part of the β-tubulin gene, and the histone H3 gene, respectively. Sequences of these three regions had 99, 99, and 97% similarity with references sequences of “C.” pauciseptatum (isolate Cy238; accessions ITS [JF735307]; β-tubulin [JF735435], and histone H3 [JF735582], respectively). To evaluate pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated with two isolates by immersing them in a conidial suspension (106 conidia ml–1) for 60 min. Ten single-vine replicates were used for each isolate, and 10 water-inoculated vines were included as controls. Thirty days after inoculation, vines were re-inoculated with 40 ml of a 106 conidia ml–1 suspension to ensure root infection. After 4 months, the inoculated plants had reduced root mass relative to controls (39.18% for Cy12UFSM and 18.27% for Cy13UFSM). Inoculated plants also had root and crown necrosis, vascular lesions, shoot decline, and vine mortality (60 and 80% mortality for Cy12UFSM and Cy13UFSM, respectively). All water-inoculated control plants remained symptomless. The fungi Cy12UFSM and Cy13UFSM were re-isolated from infected woody tissues, confirming Koch's postulates. To our knowledge, this is the first report of “C.” pauciseptatum associated with black foot disease of grapevine in Brazil, which may potentially impact the sustainability of grapevine nurseries and vineyard productivity. References: (1) L. R. Garrido et al. Fitopatol. Brasil. 29:548, 2004. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) H. J. Schoers et al. Mycol. Res. 112:82, 2008. (4) T. J. White et al. Amplification Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Cultivated grapevine (Vitis labrusca and V. vinifera) is of considerable economic importance to the Brazilian fruit industry for both fresh market consumption and for the production of wines, sparkling beverages, and juices. Black foot disease is caused by fungi of the genera Ilyonectria P. Chaverri & C. Salgado (anamorph: Cylindrocarpon Wollew.), Campylocarpon Halleen, Schroers & Crous, and Cylindrocladiella Boesew. In 2012, 4- to 40-year-old grapevines (Vitis spp.) showing reduced vigor, vascular lesions, necrotic root lesions, delayed budding, vine decline, and death were collected from seven locations at Rio Grande do Sul state, Brazil. Fungal isolations were made from root fragments and crown lesions (at least 2 cm above the bottom) on potato dextrose agar (PDA) medium added with 0.5 g L–1 streptomycin sulfate. Eight isolates were obtained and identified on the basis of morphological features and multi-gene analysis (rDNA-ITS, β-tubulin, and histone H3) as Ilyonectria macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado. One representative isolate (Cy5UFSM) was used for more detailed morphological and molecular characterization, and pathogenicity confirmation. When incubated in the dark at 20°C for 7 to 10 days, colonies of felty straw-colored mycelium (3) 4.79 cm diameter on average were observed. No sporodochia or other fruiting bodies were produced on carnation leaf agar (CLA) medium after 30 days. Microconidia that were produced after 5 weeks on spezieller nährstoffarmer agar (SNA) medium with addition of two pieces of 1 cm2 filter paper showed ovoid and ellipsoid shape (6.4 × 3.6 μm) and one-septate macroconidia (17.3 × 4.1 μm). To confirm the species, primer pairs ITS1 and ITS4 (4); Bt2a and Bt2b; and H3-1a and H3-1b (2) were used to amplify the ITS1-5.8S rRNA-ITS2, part of the β-tubulin and histone H3 genes, respectively. Sequences of these three regions showed 99, 100, and 100% of homology with I. macrodidyma, respectively. To confirm pathogenicity, 4-month-old rooted cuttings of V. labrusca cv. Bordô were inoculated by immersing them in a conidial suspension of the isolate (106 conidia ml–1) for 60 min (1). Thirty days later, inoculation was performed again by drenching the crown with 40 ml of 106 conidia ml–1 suspension to ensure infection of the roots. In the control treatment, plants were inoculated with sterile distilled water. Plants inoculated with I. macrodidyma showed necrosis of the leaf ribs, reduction in root mass, root and crown necrosis, browning of vessels, drying of shoots, and death. I. macrodidyma was re-isolated from the crown necrosis and vascular lesions, confirming Koch's postulates. To our knowledge, this is the first report of I. macrodidyma associated with black foot disease of grapevine in Brazil, which poses considerable threat to the industry unless management options are realized. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 2012. (2) N. L. Glass et al. Appl. Environ. Microbiol. 61:1323, 1995. (3) R. W. Rayner. A Mycological Colour Chart. Commonwealth Mycological Institute and British Mycological Society, 1970. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

Black foot disease is a common and destructive root disease of grapevine caused by a multitude of cylindrocarpon-like fungi in many viticultural areas of the world. This study identified 12 cylindrocarpon-like fungal species across five genera associated with black foot disease of grapevine and other diverse root diseases of fruit and nut crops in the Central Valley Region of California. Morphological observations paired with multi-locus sequence typing of four loci, internal transcribed spacer region of nuclear rDNA ITS1–5.8S–ITS2 (ITS), beta-tubulin (TUB2), translation elongation factor 1-alpha (TEF1), and histone (HIS), revealed 10 previously described species; Campylocarpon fasciculare, Dactylonectria alcacerensis, D. ecuadoriensis, D. macrodidyma, D. novozelandica, D. torresensis, D. valentina, Ilyonectria capensis, I. liriodendri, I. robusta, and two new species, Neonectria californica sp. nov., and Thelonectria aurea sp. nov. Phylogenetic analyses of the ITS+TUB2+TEF1 combined dataset, a commonly employed dataset used to identify filamentous ascomycete fungi, was unable to assign some species, with significant support, in the genus Dactylonectria, while all other species in other genera were confidently identified. The HIS marker was essential either singly or in conjunction with the aforementioned genes for accurate identification of most Dactylonectria species. Results from isolations of diseased plant tissues revealed potential new host associations for almost all fungi recovered in this study. This work is the basis for future studies on the epidemiology and biology of these important and destructive plant pathogens.

Replant disease refers to the poor growth of trees when attempting to establish the same or related species on old orchard sites. The use of pre-plant Brassicaceae seed meal (SM) soil amendments in combination with apple replant disease-tolerant rootstock genotypes has been shown to be a promising strategy for the control of apple replant disease (ARD). However, optimizing microorganism-driven protection of apple roots from infection by multiple soil-borne pathogens requires a more comprehensive understanding of how “effective” vs. “ineffective” Brassicaceae seed meal × rootstock genotype disease control systems modulate the composition of rhizosphere microbial communities. In particular, the community of oomycetes associated with the apple rhizosphere remains relatively unexplored compared with bacteria and fungi. To address these issues, we sequenced the root associated bacterial, fungal, and oomycete communities of apple replant disease tolerant (G.210) and susceptible (M.26) rootstocks when grown in an orchard replant soil amended with different Brassicaceae seed meal formulations (Brassica juncea + Sinapis alba, B. juncea, and Brassica napus) previously shown to provide varying levels of replant disease control. Multiple microbial components were associated with observed growth differences between “effective” and “ineffective” disease control systems including the absolute abundance of Ilyonectria/Cylindrocarpon in fine root tissue. Amplicon sequencing provided a more detailed picture of the genetic diversity of oomycete groups in the apple rhizosphere than previously appreciated, and highlighted the variability in oomycete community structure between different rootstock × seed meal disease control systems. In Brassica juncea + Sinapis alba SM-structured rhizospheres, the ARD-tolerant rootstock (G.210) harbored higher relative abundances of Peronosporales with reduced potential to infect apple roots and incite replant disease (such as Peronospora destructor and P. acanthicum), whereas the Peronosporales community associated with the sensitive rootstock (M.26) was dominated by the ARD-specific pathogen Phytophthora cactorum. In addition, Brassica juncea + Sinapis alba SM-structured microbiomes were characterized by numerous bacterial and fungal taxa with the potential for biocontrol, biodegradation and bioremediation. Taken together, these results support the hypothesis that particular Brassicaceae SM soil amendments not only provide “effective” disease control, but also promote microbiomes which are likely to contribute to long-term orchard soil health in many other ways. Overall, this comprehensive analysis highlights the significance of the rootstock × seed meal interaction on bacterial, fungal, and oomycete communities within the apple rhizosphere of “effective” vs. “ineffective” disease control systems and the potential influence of these elements on the dynamics of apple replant disease.

Paris polyphylla var. yunnanensis is an important medicinal plant with abundant saponins that are widely used in the pharmaceuticals industry. It is unclear why the levels of active ingredients increase as these plants age. We speculated that the concentrations of those components in the rhizomes are mediated by fungal endophytes. To test this hypothesis, we took both culture-dependent and -independent (metagenomics) approaches to analyze the communities of endophytic fungi that inhabit those rhizomes in plants of different age classes (four, six, and eight years old). In all, 147 isolates representing 18 fungal taxa were obtained from 270 segments (90 per age class). Based on morphological and genetic characteristics, Fusarium oxysporum (46.55% frequency of occurrence) was the predominant endophyte, followed by Leptodontidium sp. (8.66%) and Trichoderma viride (6.81%). Colonization of endophytic fungi was maximized in the eight-year-old rhizomes (33.33%) when compared with four-year-old (21.21%) and six-year-old (15.15%) rhizomes. Certain fungal species were present only at particular ages. For example, Alternaria sp., Cylindrocarpon sp., Chaetomium sp., Paraphaeosphaeria sporulosa, Pyrenochaeta sp., Penicillium swiecickii, T. viride, and Truncatella angustata were found only in the oldest plants. Analysis of (metagenomics) community DNA extracted from different-aged samples revealed that, at the class level, the majority of fungi had the highest sequence similarity to members of Sordariomycetes, followed by Eurotiomycetes and Saccharomycetes. These results were mostly in accord with those we obtained using culture methods. Fungal diversity and richness also changed over time. Our investigation is the first to show that the diversity of fungi in rhizomes of P. polyphylla var. yunnanensis is altered as plants age, and our findings provide a foundation for future examinations of useful compounds.

Summary Cylindrocarpon-like fungi are globally distributed plant pathogens and have a wide range of host species. However, very little is known about the species that live in the topsoil of forests and their potential role in reducing the natural regeneration of tree species, particularly of forest trees that produce abundant fruit only once every few years. To enhance our understanding of the species that inhabit the topsoil, we studied the diversity and pathogenicity of Cylindrocarpon-like fungi inhabiting the litter in old-growth mixed-beech forests in the Carpathians (Poland) and in the Alps (Austria), and in a managed beech stand in the Krakowsko-Czestochowska Highland (Poland). The fungi inhabiting the beech litter were investigated using beechnuts and pine seedlings as bait. Isolates were identified based on morphology and DNA sequencing. The pathogenicity of the most common species was investigated by inoculating beech germinants. A wide range of Cylindrocarpon-like fungi were associated with the beech litter: 718 cultures representing 12 species were isolated. Five taxa were identified down to species level: namely Ilyonectria crassa, I. pseudodestructans, I. rufa, Neonectria candida and N. obtusispora, and seven species were identified to genus level (Neonectria or Ilyonectria species). Ilyonectria destructans, which is considered to be the sexual morph of ‘Cylindrocarpon destructans’, was not found. There were qualitative and quantitative differences between the different forest sites in terms of Neonectria and Ilyonectria species composition in beech litter. The isolation frequency and species richness of Cylindrocarpon-like fungi were greatest in beech litter taken from old-growth mixed-beech forests. Neonectria and Ilyonectria species were capable of killing beech germinants, suggesting that they may play a negative role in natural beech regeneration.

Background: Root diseases caused by Cylindrocarpon destructans and Fusarium solani decrease the yield and quality of ginseng. Cylindrocarpon root rot is a major disease caused by replant failure in ginseng fields. Methods and Results: Solarization of infested greenhouse soil was carried out during the summer season after applying green manure (Sudan grass) and Calcium Cyanamide (CC) on the soil. Mycelium and conidia of C. destructans died at after 15 h, but they did not die at after 15 h. They also died after keeping the soil at for 2 h daily for 9 days, and at for 8 days, but they did not die at for 9 days. Maximum soil temperature was at 5 cm depth, at 10 cm, at 15 cm, at 20 cm, and at 30 cm by incorporating green manure into the soil and using solarization. Solarization using green manure mixed with CC was the most effective in decreasing soil-borne pathogens of 2-year-old ginseng. However, the addition of CC decreased the root weight due to the increase in EC and . Conclusions: Soil disinfection using green manure and solarization in a greenhouse environment was effective in inhibiting root rot, however, it did not completely kill the soil-borne pathogens.

The genus Thelonectria and closely related species with cylindrocarpon-like asexual states are a group of perithecial ascomycetes in the family Nectriaceae that occur as saprobes and in few cases as pathogens of hardwood trees, shrubs or other plants. Although a key component of forest ecosystems around the world, species relationships and distributions of these fungi are largely unknown. The objectives of this study were to: 1) infer species rank phylogenetic relationships of the genus Thelonectria and closely related species with cylindrocarpon-like asexual states and test the monophyly of each of the groups studied; 2) delimit taxa establishing taxon circumscriptions; 3) resolve nomenclatural issues by identifying redundantly used names and synonyms; and 4) provide an updated outline to the genus, geographical distributions data and identification tools, specifically diagnostic keys and molecular data that can be used as molecular barcodes. The recovered consensus phylogeny resulted in a narrow circumscription of the genus Thelonectria, based on the type T. discophora, excluding one of the common species T. jungneri. According to the phylogenetic analyses, T. jungneri belongs in a segregate clade that should be recognized as a different genus. In the genus Thelonectria, a total of four new species and three new combinations are recognized. Additionally, three new genera, closely related to Thelonectria, are described to accommodate species displaying a morphological resemblance to those of Thelonectria: Cinnamomeonectria gen. nov. with C. cinnamomea as type species, Macronectria gen. nov. with M. jungneri as type species and including four additional newly described species, and Tumenectria gen. nov. with T. laetidisca as type species.

Atractylodes lancea is a type of typical traditional Chinese medicinal (TCM) herb that is economically important in China. The traditional planting method of A. lancea is to plant in situ continuously for many years, which often leads to impediments for its growth and development and soil-borne diseases. The root-associated microbiome is believed to play an important role in plant resistance and the quality of products from the plant. This study aims to reveal detailed changes in the populations of rhizosphere microorganisms, and providing theoretical guidance for the prevention and control of soil-borne diseases in A. lancea. A high-throughput sequencing approach was utilized to illustrate changes in the microbial community from different planting years. Results and conclusions: The results show that the diversity and composition of the root-associated microbiome was significantly impacted by the consecutive monoculture of A. lancea. At the level of the comparisons of the phyla, Bacteroidetes, Proteobacteria, Ascomycota, and Basidiomycota declined significantly. In contrast, the relative abundance of Actinobacteria, Acidobacteria, and Mortierellomycota distinctly increased. Comparisons at the genus level indicated that Sphingomonas, Flavobacterium, Pseudomonas, Pedobacter, and Tausonia decreased significantly, whereas Mortierella, Cylindrocarpon, Dactylonectria, and Mucor distinctly increased. In conclusion, this study helps to develop an understanding of the impediments involved in the consecutive monoculture of A. lancea.