F. Peduto

Characterization of fungal pathogens associated with grapevine trunk diseases in Arkansas and Missouri

Grapevine trunk diseases are a major concern to the wine- and table-grape industries worldwide, limiting both vineyard longevity and productivity. Field surveys conducted throughout the grape-growing regions of Arkansas and Missouri revealed the presence of three economically important grapevine trunk diseases including, Botryosphaeria canker, Eutypa dieback and esca. Morphological studies along with multi-gene phylogenetical analyses confirmed the identification of 15 different fungal taxa associated with different vascular symptoms. These include Botryosphaeria dothidea, Diatrypella sp., Diplodia seriata, Dothiorella americana, Eutypa lata, Eutypella vitis, Lasiodiplodia missouriana, Lasiodiplodia viticola, Neofusicoccum ribis, Neofusicoccum vitifusiforme, Pestalotiopsis sp., Pestalotiopsis uvicola, Phaeomoniella chlamydospora, Phomopsis viticola, Schyzophyllum commune, and Togninia minima. All of these represent new records on grapevines in Arkansas and Missouri. Dothiorella americana, L. missouriana and L. viticola are described as new species, and both N. ribis and N. vitifusiforme are first reported as grapevine pathogens in North America. Koch’s postulates confirmed the pathogenicity of all fungal species except S. commune in the interspecific hybrids Vignoles, Chambourcin, Norton, and Traminette. Lasiodiplodia spp., N. ribis, and P. viticola were the most virulent fungi, while D. americana, E. vitis and N. vitifusiforme were considered to be weak pathogens. This research highlights the importance that grapevine trunk diseases have on grapevine health in growing regions where, due to different climatological conditions, interspecific hybrid cultivars are predominantly grown.

Olive Twig and Branch Dieback: Etiology, Incidence, and Distribution in California

Eighteen different fungal species were isolated from symptomatic wood of olive trees (Olea europaea) affected by twig and branch dieback in California and identified by means of morphological characters and multigene sequence analyses of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2), a partial sequence of the β-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). These species included Diaporthe viticola, Diatrype oregonensis, Diatrype stigma, Diplodia mutila, Dothiorella iberica, Lasiodiplodia theobromae, Phaeomoniella chlamydospora, Phomopsis sp. group 1, Phomopsis sp. group 2, and Schizophyllum commune, which are for the first time reported to occur in olive trees; Eutypa lata, Neofusicoccum luteum, Neofusicoccum vitifusiforme, and Phaeoacremonium aleophilum, which are for the first time reported to occur in olive trees in the United States; and Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum mediterraneum, and Trametes versicolor, which have been previously reported in olive trees in California. Pathogenicity studies conducted in olive cultivars Manzanillo and Sevillano showed N. mediterraneum and Diplodia mutila to be the most virulent species and Diatrype stigma and D. oregonensis the least virulent when inoculated in olive branches. Intermediate virulence was shown for the rest of the taxa. This study demystifies the cause of olive twig and branch dieback and elucidates most of the fungal pathogens responsible for this disease in California.

Taxonomy and DNA phylogeny of Diatrypaceae associated with Vitis vinifera and other woody plants in Australia

The Diatrypaceae occur worldwide and comprise a number of pathogens of woody crops, forest and ornamental tree species. Despite the taxonomic difficulties within this family, interest in the Diatrypaceae has increased recently, mainly due to the recent detection of these fungi in the premium grape growing regions of California. In the present study, we investigated the diversity and host range of diatrypaceous fungi from prominent wine grape growing regions in South Australia, New South Wales and Western Australia. Approximately 100 isolates were collected from grapevine and other woody plants and compared with reference collections from the United States and Europe. Phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA and partial sequence of the β-tubulin gene, combined with morphological analyses separated 12 species. These included the previously described species Cryptovalsa ampelina, C. rabenhorstii, Diatrype brunneospora, Eutypa lata, E. leptoplaca, Eutypella australiensis, E. citricola, a Cryptosphaeria sp. and a Diatrype sp., whereas Diatrypella vulgaris, Eutypella cryptovalsoidea and E. microtheca are described as new. Seven species were isolated from grapevine but the prevalence of Diatrypaceae in grapevine cankers varied among the regions surveyed. In many instances in WA and NSW, these newly reported fungi were more widespread and abundant than E. lata. This study provides new information to assist with diagnosis of the causal agents of dieback and canker diseases in Australia and development of management strategies. Further studies to characterize the pathogenicity of diatrypaceous species to grapevines and to elucidate the biology of these fungi are underway.

First report of Diplodia corticola causing grapevine (Vitis vinifera) cankers and trunk cankers and dieback of Canyon live oak (Quercus chrysolepis) in California.

The botryosphaeriaceous fungus Diplodia corticola A. J. L. Phillips, Alves & Luque was shown to be the most prevalent canker and dieback pathogen in cork oaks (Quercus suber L.) in the Iberian Peninsula causing a general decline of the trees as a consequence of canker formation in the trunks (1). In addition, D. corticola has been recently reported as a grapevine pathogen causing cankers in the vascular tissue of 1-year-old canes, spurs, and cordons in Texas (3). In 1998, Jacobs and Rehner reported one isolate of D. corticola from oak in California, but no information regarding the oak species from which the isolate was obtained and its virulence were available in the study (2). In 2009, D. corticola was isolated on potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride from symptomatic grapevine cordons and on acidified PDA from the trunk of a canyon live oak tree from Sonoma and Plumas counties, respectively. Two grapevine isolates (UCD1260So and UCD1275So) and one oak isolate (CDFA519) were examined and morphologically compared with previously identified D. corticola isolates CBS678.88 and UCD2397TX from cork oak from Spain and grapevine in Texas, respectively. D. corticola colonies from California were characterized by moderately fast-growing, dark olivaceous, and dense aerial mycelium on PDA. Conidia were obtained from pycnidia formed on pine needles placed on 2% water agar. Conidia were hyaline, contents granular, aseptate, thick walled, ellipsoidal, sometimes becoming dark brown and septate with age. Nucleotide sequences of three genes (ITS1-5.8S-ITS2, a partial sequence of the beta-tubulin gene BT2, and part of the translation elongation factor EF1-α) from D. corticola isolates UCD1260So, UCD1275So, and CDFA519 from California were amplified. All DNA sequences from grapevine and oak tree isolates from California showed 99 to 100% homology with D. corticola isolates previously identified and deposited into GenBank. All DNA sequences obtained from Californian isolates were also deposited into GenBank. Pathogenicity tests were conducted by inoculating detached Vitis vinifera cv. Red Globe dormant canes and canyon live oak branches with agar plugs of isolates UCD1260So, UCD1275So, and CDFA519 (10 inoculations per isolate per host) as described by Úrbez-Torres et al. (3). The same number of grapevine canes and oak branches were inoculated with noncolonized agar plugs as controls. Six weeks after inoculation, the extent of vascular discoloration that developed from the point of inoculation was measured. D. corticola isolates UCD1260So, UCD1275So, and CDFA519 caused an average vascular lesion length of 30.4, 29.6, and 24 mm and 15, 13.2, and 8.6 mm in grapevine dormant canes and oak branches, respectively. Furthermore, D. corticola isolates from grapevine were pathogenic in oak branches and vice versa. Reisolation of D. corticola from discolored vascular tissue of inoculated material was 100%. The extent of vascular discoloration from inoculated grapevine canes and oak branches was significantly greater (P < 0.05) compared with the controls (1.8 and 2 mm, respectively). No fungi were reisolated from the slightly discolored tissue of the controls. To our knowledge, this is the first report of D. corticola causing grapevine cankers and oak trunk cankers in California. References: (1) A. Alves et al. Mycologia 96:598, 2004. (2) K. A. Jacobs and S. A. Rehner. Mycologia 90:601, 1998. (3) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

First report of grapevine cankers caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California.

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8-) 27.5 to 30.5 (-33.4) × (12.1) 14.3 to 16.8 (-18.2) μm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2-) 20.5 to 27.8 (-29) × (5.1) 5.9 to 6.5 (-7.2) μm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukeys test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California. References: (1) T. I. Burgess et al. Mycologia 98:423, 2006. (2) P. W. Crous et al. Fungal Planet. No. 19, 2007. (3) J. R. Úrbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009. (4) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

Some Observations on the Relationship of Manifest and Hidden Esca to Rainfall

Summary. This paper reports observations on the relationship between the yearly incidence of manifest esca (i.e. diseased plants which show foliar symptoms), hidden esca (that which remains asymptomatic throughout a growing season) and rainfall. Data from three vineyards (two in Tuscany and one in Emilia-Romagna, Italy) showed that rainfall in May–July or only in July was inversely related with hidden esca. For two vineyards, TB in Emilia-Romagna and CAR-3 in Tuscany, the spatial pattern of diseased vines in the first year of appearance of the foliar esca symptoms was also determined. The maps of the vines in these vineyards indicated that diseased plants mostly occurred alone. This suggests that the disease had its origin in infected rooted cuttings or was triggered by inoculum aerially dispersed from distant sources and/or occurring, at least in hypothesis, in the soil.

First Report of Twig and Branch Dieback of English Walnut (Juglans regia) Caused by Neofusicoccum mediterraneum in California

California produces 99% of the U.S. English walnut crop with more than 30 cultivars on ~89,000 ha. Production for 2008 was ~436,000 tons with a value of

Effect of High Temperature and Exposure Time on Erysiphe necator Growth and Reproduction: Revisions to the UC Davis Powdery Mildew Risk Index

527 million. In early summer of 2009 and 2010, branch and twig dieback of English walnut (Juglans regia L.) was detected in orchards in Yolo County and submitted to our diagnostic laboratory. Disease symptoms included death of twig tips, branch dieback, wood lesions, and canker formation. Pycnidia were embedded within the bark of dead twigs. Conidia from pycnidia were hyaline, fusoid-ellipsoidal, widest usually in the middle, and 21 to 24 (-27) × 5 to 7 μm (n = 30). Isolations from cankers yielded the fungus Neofusicoccum mediterraneum Crous, M.J. Wingf. & A.J.L. Phillips (1). Fungal colonies of N. mediterraneum grew light olive green to gray on potato dextrose agar, becoming dark olive green with age. Identification of fungal isolates was confirmed by sequence comparison of Californian isolates with ex-type (CBS 121558) sequences in GenBank (3) using the internal transcribed spacer region of the rDNA, a portion of the β-tubulin gene, and part of the translation elongation factor. Sequences of Californian isolates (GenBank HM443604-HM443609) were identical to the ex-type sequences for all three genes. Previous studies in California reported the occurrence and pathogenicity of N. mediterraneum into grapevine (Vitis vinifera L.) (3) and almond trees (Prunis dulcis L.) (2). Inderbitzin et al (2) investigated the host range of N. mediterraneum in California and reported the occurrence of pycnidia on English walnut trees. However, this study did not investigate the pathogenicity of N. mediterraneum on this host. In the current study, the pathogenicity of N. mediterraneum in J. regia cvs. Hartley and Chandler was investigated in an orchard at UC Davis using two fungal isolates. Pathogenicity tests were performed by inoculating eight 2- to 4-year-old branches of mature J. regia trees. Inoculations were made in June 2009 with a 5-mm cork borer to remove bark and placing an 8-day-old 5-mm-diameter agar plug bearing fresh mycelium of the fungal isolates directly into the fresh wound, mycelium side down. An additional eight branches of each cultivar were inoculated with noncolonized agar plugs to serve as controls. Inoculated wounds were covered with petroleum jelly and wrapped with Parafilm to retain moisture. Branches were harvested after 10 months of incubation and checked for canker development. The extent of vascular discoloration was measured in each branch and isolations were made from the edge of discolored tissue to confirm Kochs postulates. Statistical analyses were performed with analysis of variance and Dunnetts t-test to assess significant differences in the extent of vascular discoloration between inoculations with N. mediterraneum and the control. Necrosis length for the two isolates averaged 131.5 mm in Hartley branches and 110 mm in the Chandler branches. Average necrosis lengths in the control branches were 18.5 mm and 16.7 mm, respectively, significantly lower (P < 0.05) than the average necrosis length found in branches inoculated with N. mediterraneum. Fungal recovery was 75% in both varieties. To our knowledge, this study is the first report of N. mediterraneum as a pathogen of J. regia trees in California. References: (1) P. W. Crous et al. Fungal Planet 19, 2007. (2) P. Inderbitzin et al. Mycologia. Online publication. doi:10.3852/10-006, 2010. (3) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

First report of Ilyonectria macrodidyma causing root rot of olive trees (Olea europaea) in California.

Epidemics of powdery mildew, the most important disease of grapevines in California, are driven by moderate temperatures (21 to 30°C). High temperatures can delay or stop the growth and sporulation of Erysiphe necator. Using controlled conditions, we investigated the response of the pathogens colony growth, conidiospore production, and germination to eight temperatures (30 to 44°C) at 12 exposure times (0.25 to 24 h). The pathogen survived, grew, and reproduced at higher temperatures than previously reported; exposure time was as important as temperature in defining lethal and sublethal effects. Lethal effects started at 36 to 38°C. Lethal exposure times decreased with increasing temperature. Based on this new information on the pathogens biology, we tested revisions to the high-temperature threshold of the UC Davis Powdery Mildew Risk Index in field settings. No differences in the total number of fungicide applications resulted from the use of either the original or the revised models. However, use of the 38°C for 2 h threshold consistently showed equal (leaves) or better (fruit) disease control compared with the original model, as well as equal disease control compared with a calendar schedule, but with five fewer applications over the 2 years of the study.

First report of Calosphaeria pulchella associated with branch dieback of sweet cherry trees in California

The California olive industry produces 99% of the U.S. olive crop, which represented a value of over