Genera of phytopathogenic fungi: GOPHY 4.

This paper is the fourth contribution in the Genera of Phytopathogenic Fungi (GOPHY) series. The series provides morphological descriptions and information about the pathology, distribution, hosts and disease symptoms, as well as DNA barcodes for the taxa covered. Moreover, 12 whole-genome sequences for the type or new species in the treated genera are provided. The fourth paper in the GOPHY series covers 19 genera of phytopathogenic fungi and their relatives, including Ascochyta, Cadophora, Celoporthe, Cercospora, Coleophoma, Cytospora, Dendrostoma, Didymella, Endothia, Heterophaeomoniella, Leptosphaerulina, Melampsora, Nigrospora, Pezicula, Phaeomoniella, Pseudocercospora, Pteridopassalora, Zymoseptoria, and one genus of oomycetes, Phytophthora. This study includes two new genera, 30 new species, five new combinations, and 43 typifications of older names. Taxonomic novelties: New genera: Heterophaeomoniella L. Mostert, C.F.J. Spies, Halleen & Gramaje, Pteridopassalora C. Nakash. & Crous; New species: Ascochyta flava Qian Chen & L. Cai, Cadophora domestica L. Mostert, R. van der Merwe, Halleen & Gramaje, Cadophora rotunda L. Mostert, R. van der Merwe, Halleen & Gramaje, Cadophora vinacea J.R. Úrbez-Torres, D.T. O'Gorman & Gramaje, Cadophora vivarii L. Mostert, Havenga, Halleen & Gramaje, Celoporthe foliorum H. Suzuki, Marinc. & M.J. Wingf., Cercospora alyssopsidis M. Bakhshi, Zare & Crous, Dendrostoma elaeocarpi C.M. Tian & Q. Yang, Didymella chlamydospora Qian Chen & L. Cai, Didymella gei Qian Chen & L. Cai, Didymella ligulariae Qian Chen & L. Cai, Didymella qilianensis Qian Chen & L. Cai, Didymella uniseptata Qian Chen & L. Cai, Endothia cerciana W. Wang. & S.F. Chen, Leptosphaerulina miscanthi Qian Chen & L. Cai, Nigrospora covidalis M. Raza, Qian Chen & L. Cai, Nigrospora globospora M. Raza, Qian Chen & L. Cai, Nigrospora philosophiae-doctoris M. Raza, Qian Chen & L. Cai, Phytophthora transitoria I. Milenkovic, T. Májek & T. Jung, Phytophthora panamensis T. Jung, Y. Balci, K. Broders & I. Milenkovic, Phytophthora variabilis T. Jung, M. Horta Jung & I. Milenkovic, Pseudocercospora delonicicola C. Nakash., L. Suhaizan & I. Nurul Faziha, Pseudocercospora farfugii C. Nakash., I. Araki, & Ai Ito, Pseudocercospora hardenbergiae Crous & C. Nakash., Pseudocercospora kenyirana C. Nakash., L. Suhaizan & I. Nurul Faziha, Pseudocercospora perrottetiae Crous, C. Nakash. & C.Y. Chen, Pseudocercospora platyceriicola C. Nakash., Y. Hatt, L. Suhaizan & I. Nurul Faziha, Pseudocercospora stemonicola C. Nakash., Y. Hatt., L. Suhaizan & I. Nurul Faziha, Pseudocercospora terengganuensis C. Nakash., Y. Hatt., L. Suhaizan & I. Nurul Faziha, Pseudocercospora xenopunicae Crous & C. Nakash.; New combinations: Heterophaeomoniella pinifoliorum (Hyang B. Lee et al.) L. Mostert, C.F.J. Spies, Halleen & Gramaje, Pseudocercospora pruni-grayanae (Sawada) C. Nakash. & Motohashi., Pseudocercospora togashiana (K. Ito & Tak. Kobay.) C. Nakash. & Tak. Kobay., Pteridopassalora nephrolepidicola (Crous & R.G. Shivas) C. Nakash. & Crous, Pteridopassalora lygodii (Goh & W.H. Hsieh) C. Nakash. & Crous; Typification: Epitypification: Botrytis infestans Mont., Cercospora abeliae Katsuki, Cercospora ceratoniae Pat. & Trab., Cercospora cladrastidis Jacz., Cercospora cryptomeriicola Sawada, Cercospora dalbergiae S.H. Sun, Cercospora ebulicola W. Yamam., Cercospora formosana W. Yamam., Cercospora fukuii W. Yamam., Cercospora glochidionis Sawada, Cercospora ixorana J.M. Yen & Lim, Cercospora liquidambaricola J.M. Yen, Cercospora pancratii Ellis & Everh., Cercospora pini-densiflorae Hori & Nambu, Cercospora profusa Syd. & P. Syd., Cercospora pyracanthae Katsuki, Cercospora horiana Togashi & Katsuki, Cercospora tabernaemontanae Syd. & P. Syd., Cercospora trinidadensis F. Stevens & Solheim, Melampsora laricis-urbanianae Tak. Matsumoto, Melampsora salicis-cupularis Wang, Phaeoisariopsis pruni-grayanae Sawada, Pseudocercospora angiopteridis Goh & W.H. Hsieh, Pseudocercospora basitruncata Crous, Pseudocercospora boehmeriigena U. Braun, Pseudocercospora coprosmae U. Braun & C.F. Hill, Pseudocercospora cratevicola C. Nakash. & U. Braun, Pseudocercospora cymbidiicola U. Braun & C.F. Hill, Pseudocercospora dodonaeae Boesew., Pseudocercospora euphorbiacearum U. Braun, Pseudocercospora lygodii Goh & W.H. Hsieh, Pseudocercospora metrosideri U. Braun, Pseudocercospora paraexosporioides C. Nakash. & U. Braun, Pseudocercospora symploci Katsuki & Tak. Kobay. ex U. Braun & Crous, Septogloeum punctatum Wakef.; Neotypification: Cercospora aleuritis I. Miyake; Lectotypification: Cercospora dalbergiae S.H. Sun, Cercospora formosana W. Yamam., Cercospora fukuii W. Yamam., Cercospora glochidionis Sawada, Cercospora profusa Syd. & P. Syd., Melampsora laricis-urbanianae Tak. Matsumoto, Phaeoisariopsis pruni-grayanae Sawada, Pseudocercospora symploci Katsuki & Tak. Kobay. ex U. Braun & Crous. Citation: Chen Q, Bakhshi M, Balci Y, Broders KD, Cheewangkoon R, Chen SF, Fan XL, Gramaje D, Halleen F, Horta Jung M, Jiang N, Jung T, Májek T, Marincowitz S, Milenkovic T, Mostert L, Nakashima C, Nurul Faziha I, Pan M, Raza M, Scanu B, Spies CFJ, Suhaizan L, Suzuki H, Tian CM, Tomsovský M, Úrbez-Torres JR, Wang W, Wingfield BD, Wingfield MJ, Yang Q, Yang X, Zare R, Zhao P, Groenewald JZ, Cai L, Crous PW (2022). Genera of phytopathogenic fungi: GOPHY 4. Studies in Mycology 101: 417-564. doi: 10.3114/sim.2022.101.06.

Epidemiology and Genetic Diversity of Grapevine Leafroll-Associated Viruses in British Columbia.

Grapevine leafroll disease (GLD) is a complex associated with one or more virus species belonging to the family Closteroviridae. The majority of viruses in this complex are vectored by one or more species of mealybugs (Pseudococcidae) and/or scale insects (Coccidae). Grape-growing regions of British Columbia (BC), including Okanagan, Similkameen, and Fraser valleys and Kamloops (BC central interior), Vancouver, and Gulf islands, were surveyed during the 2014 and 2015 growing seasons for the presence of four major grapevine leafroll-associated viruses, including Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4. In total, 3,056 composite five-vine samples were collected from 153 Vitis vinifera and three interspecific hybrid vineyard blocks. The results showed GLRaV-3 to be the most widespread, occurring in 16.7% of the composite samples, followed by GLRaV-4 (3.9%), GLRaV-1 (3.8%), and GLRaV-2 (3.0%). Mixed infections of two or more GLRaVs were found in 4.1% of the total samples. The relative incidence of GLRaVs differed among regions and vineyard blocks of a different age. Characterization of partial CO1 region from a total of 241 insect specimens revealed the presence of Pseudococcus maritimus, Parthenolecanium corni, and other Pulvinaria sp. in BC vineyards. Spatial patterns of GLRaV-3 infected grapevines in three vineyard blocks from three different regions in the Okanagan Valley showed variable degrees of increase in disease spread ranging from 0 to 19.4% over three growing seasons. Regional differences in the relative incidence and spread of GLD underline the need for region-based management programs for BC vineyards.

Botryosphaeriaceae Species Associated With Cankers and Dieback Symptoms of Acacia mangium and Pinus caribaea var. hondurensis in Venezuela.

Several species in the Botryosphaeriaceae family cause wood stain, cankers, and dieback of trunks and branches in a wide range of forest tree species. The aim of this study was to characterize the botryosphaeriaceous fungi associated with decline symptoms observed in Acacia mangium and Pinus caribaea var. hondurensis, two economically important forest tree species grown in commercial plantations in Venezuela. Fungi isolated from symptomatic samples collected from both hosts in commercial sites were identified based on their morphology and DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2) and part of the ß-tubulin and translation elongation factor 1-α genes. Lasiodiplodia theobromae and L. venezuelensis were routinely isolated from A. mangium and P. caribaea var. hondurensis. Additionally, the novel species Diplodia guayanensis was isolated and characterized from symptomatic and asymptomatic tissues of A. mangium. Multigene phylogenetic analyses along with restriction fragment length polymorphism studies further supported the identification of these species. A pathogenicity study was conducted under natural conditions and 12 weeks after inoculation all Botryosphaeriaceae spp. were shown to be highly virulent on A. mangium. Contrary, no lesions were observed in the wood of P. caribaea var. hondurensis when inoculated with L. theobromae and L. venezuelensis. However, both species were consistently reisolated from the asymptomatic tissue beyond the inoculation point. This study contributes to a better understand the role that species in the Botryosphaeriaceae play on disease symptoms and dieback of A. mangium and P. caribaea var. hondurensis from plantations in eastern Venezuela.

Development of a DNA Macroarray for the Detection and Identification of Fungal Pathogens Causing Decline of Young Grapevines.

Young vine decline (YVD) is a complex disease caused by at least 51 different fungi and responsible for important economic losses to the grapevine industry worldwide. YVD fungi are known to occur in planting material. Hence, detection prior to planting is critical to assure longevity of newly established vineyards. A DNA macroarray based on reverse dot-blot hybridization containing 102 oligonucleotides complementary to portions of the ß-tubulin region was developed for detection of YVD fungi. Specificity of the array was first evaluated against 138 pure fungal cultures representing 72 different taxa from nine genera, including 37 YVD species. In total, 61 species, including 34 YVD pathogens, were detected and identified by the array. The detection limit of the array was below 0.1 pg of genomic DNA. The array was validated against artificially inoculated canes and soil and commercial planting material, with the latter showing a high incidence of YVD fungi in nursery plants otherwise not detected by traditional plating and culturing. This DNA array proved to be a rapid and specific tool to simultaneously detect and identify most YVD fungi in a single test, which has the potential to be used in commercial diagnostics or by the grapevine nursery industry to determine the health status of the planting material.

First Report of Lasiodiplodia pseudotheobromae Causing Trunk Cankers in Acacia mangium in Venezuela.

In May 2010, canker and wood stain symptoms in trunks and stems of 125 Acacia mangium were observed during a survey conducted in the Uverito plantations, Monagas State, Venezuela. Cankers were 20 to 65 cm long and were brownish on the margins and dark brown in the center. Many of the cankers had swollen margins and in some cases a black exudate could be seen leaking from the most severe cankers. Small pieces (4 to 5 mm) of necrotic tissues from the cankers were surface sterilized for 1 min in 1.5% NaOCl, washed twice with sterile distilled water, and plated onto potato dextrose agar amended with 0.01% tetracycline hydrochloride (PDA-tet). Plates were incubated at 25°C under near-UV light. Colonies developed from symptomatic tissue and formed a compact mycelium, which was initially white, but became dark gray with age. Based on colony morphology, isolates were tentatively identified as a member of the Botryosphaeriaceae family. Pycnidia were produced on sterilized pine needles on 2% water agar after 5 weeks of incubation at 25°C under continuous near-UV light. Conidia were ellipsoidal, initially hyaline, unicellular, becoming dark brown, and developing a thick wall, a central septum, and longitudinal striations with age. Conidia measured 26 to 31 µm long and 11 to 16 µm wide (n = 60). The conidial morphology matched that of Lasiodiplodia, a member of the Botryosphaeriaceae family (1). Primers ITS4/ITS5, Bt2a/Bt2b, and EF1-688F/EF1-1251R (2) were used to amplify and subsequently sequence the ITS1-5.8S-ITS2 region and parts of the beta-tubulin (BT) and translation elongation factor 1-alpha (TEF1-α) gene regions, respectively. The putative Lasiodiplodia isolates had 98 to 99% homology with Lasiodiplodia pseudotheobromae isolate CBS 116459 for all three loci (EF622077, EF622057, and EU673111) (1). Based on morphological characters and DNA sequencing, the canker isolates from Venezuela (CBS129752 and UCD-A1) were then identified as L. pseudotheobromae (1) and sequences were deposited in GenBank (Accession Nos. JX545091 to JX545092, JX545111 to JX545112, and JX545131 to JX545132). Pathogenicity tests were performed by inoculating 2-year-old A. mangium tree trunks with isolates CBS129752 and UCD-A1. Twenty trees per isolate were inoculated by placing a mycelium plug from the growing margin of 8-day-old colonies upside down directly into a fresh wound made with a 5-mm cork borer. Wounds were sealed with Parafilm. Ten control trees were inoculated with non-colonized PDA plugs. After 12 weeks, all inoculated seedlings showed bark swelling around the inoculation points and a brown necrosis of the wood could be observed when removing the bark. Average length necrosis above and below the point inoculation was 27.2 cm; additionally, a black exudate was observed when the outer bark was removed from inoculation points. L. pseudotheobromae was successfully reisolated from the necrotic tissue observed in symptomatic plants. No symptoms were observed in the control plants and L. pseudotheobromae was not isolated from the controls. L. pseudotheobromae has been reported in Africa, Asia, Europe, and Latin America, where it occurs on forest and fruit trees (1). This study shows L. pseudotheobromae to be highly virulent on A. mangium and, to our knowledge, this is the first report of L. pseudotheobromae on this host in Venezuela. References: (1) A. Alves et al. Fungal Diversity 28:1, 2008. (2) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006.

Grapevine Trunk Diseases in British Columbia: Incidence and Characterization of the Fungal Pathogens Associated with Black Foot Disease of Grapevine.

Black foot disease of grapevines, caused by several fungal species in the genera Campylocarpon, Cylindrocarpon, Cylindrocladiella, and Ilyonectria, causes significant economic losses to the grapevine industry worldwide. This study represents the first attempt to identify and characterize the fungal pathogens associated with black foot disease of grapevines in British Columbia (BC). Field surveys conducted throughout all grape-growing regions in BC that included assessment of foliar symptomatology and isolations from symptomatic vines showed Cylindrocarpon/Ilyonectria spp. occurred in 32 of 90 (35.5%) young vineyards surveyed (≤8 year old) and in 41 of 215 (19%) samples collected. In 20 of the 41 (48.8%) samples, Cylindrocarpon/Ilyonectria spp. were the sole fungi isolated from symptomatic tissue. In the rest of the samples, black foot fungi were found to primarily coexist with fungal taxa associated with Petri disease of grapevines. Colony and conidia phenotypical characterization, along with DNA analyses of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA, and part of the ß-tubulin and translation elongation factor 1-α genes, revealed five different black foot fungi occurring in declining young vines in BC, namely Cylindrocarpon pauciseptatum, Ilyonectria liriodendri, Ilyonectria macrodidyma, Ilyonectria robusta, and Ilyonectria torresensis. Pathogenicity studies showed all five species to be highly virulent in the grapevine rootstock cultivar 3309C. Overall, I. liriodendri and I. macrodidyma were the most virulent species when inoculated in Vitis vinifera 'Chardonnay' and rootstock 3309C.

Grapevine Trunk Diseases in British Columbia: Incidence and Characterization of the Fungal Pathogens Associated with Esca and Petri Diseases of Grapevine.

Esca and Petri disease are two economically important grapevine diseases worldwide. This study reports for the first time the occurrence of both diseases on grapevines in British Columbia (BC) and subsequently in Canada. Visual assessment of 55,699 vines in 118 vineyards revealed a low incidence of esca with only 104 (0.2%) vines showing foliar symptoms. Young vine decline (YVD) was observed in 1,910 (7.8%) of 24,487 monitored young vines and in 52 (8%) of 654 young vines used as re-plants in mature vineyards. In 8 of 51 monitored young vineyards, YVD-affected vines ranged between 15 and 55%. Morphological studies along with DNA analyses of the ITS1-5.8S-ITS2, and part of the ß-tubulin, actin, and translation elongation factor 1-α gene regions, allowed us to identify Cadophora luteoolivacea, Phaeomoniella chlamydospora, Phaeoacremonium iranianum, Togninia fraxinopennsylvanica, Togninia minima, and the novel species Phaeoacremonium canadense and Phaeoacremonium roseum from esca and Petri disease infected vines in BC. This study includes for the first time the EF1-α DNA marker in Phaeoacremonium spp. delineation. Pathogenicity studies showed all seven fungi to cause vascular symptoms similar to those observed in esca and Petri disease infected vines. Additionally, the "tiger-stripes" foliar symptom of esca was successfully reproduced when healthy potted vines were inoculated with BC isolates of Pa. chlamydospora, Pm. canadense, Pm. iranianum, T. fraxinopennsylvanica, and T. minima.

Phomopsis Dieback: A Grapevine Trunk Disease Caused by Phomopsis viticola in California.

Field surveys recently conducted in California and in other grape-growing regions in the United States showed Phomopsis viticola to be one of the most prevalent fungi isolated from grapevine perennial cankers in declining vines. The current study has not only confirmed the presence of P. viticola from grapevine cankers in California but also has for the first time revealed the occurrence of Diaporthe ambigua, D. eres, and D. neotheicola in symptomatic grapevine wood in California by means of morphological studies and multi-gene sequence analysis. Pathogenicity trials conducted on mature cordons of Vitis vinifera 'Syrah' and 'Red Globe', as well as on lignified Syrah dormant canes, showed P. viticola isolates from California to be capable of causing perennial cankers. Lengths of vascular discoloration caused by P. viticola were similar to those caused by Eutypa lata and several Botryosphaeriaceae spp., which are well-known grapevine trunk disease pathogens. Additionally, a lack of spring growth was commonly observed in dormant canes inoculated with P. viticola spore suspensions in two pathogenicity trials. As part of this study, V. vinifera 'Cabernet Sauvignon' and 'Zinfandel' wood was shown to be more susceptible to infection by P. viticola than 'Barbera', 'Chardonnay', 'Merlot', and 'Thompson Seedless' wood. After more than 40 years overlooking P. viticola as a grapevine wood pathogen, this study provides strong evidence of the role of P. viticola as a canker-causing organism, and suggests its addition to the fungi involved in the grapevine trunk disease complex. Results from this study suggest D. ambigua and D. neotheicola to be saprophytes or weak pathogens on grapevine wood.

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 ß-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.

First Report of Ilyonectria macrodidyma Causing Root Rot of Olive Trees (Olea europaea) in California.

The California olive industry produces 99% of the U.S. olive crop, which represented a value of over $113 million in 2010. During the 2008 and 2009 growing seasons, decline of young super-high-density olive cvs. Arbequina, Arbosana, and Koroneiki trees (<4 years old) was observed in orchards throughout Glenn, Yolo, and San Joaquin Counties. Symptomatic trees showed stunted growth and chlorotic leaves with roots having black, sunken, necrotic lesions, which frequently prolonged into the base and crown of the tree. Twenty-five trees were collected from different orchards and necrotic roots as well as infected trunk tissue were plated onto potato dextrose agar amended with 0.01% tetracycline hydrochloride. Cultures were incubated at room temperature (23 ± 2°C) until fungal colonies were observed. In 17 out of 25 trees collected (68%), light yellow fungal colonies were observed from the symptomatic tissue after 7 to 10 days. Colonies turned dark yellow to orange with age and showed an orange-dark brown reverse. Both microconidia (hyaline, ellipsoidal to ovoidal and aseptate (n = 60) (6.5) 11.5 to 13.5 (17.1) × (3) 3.4 to 4.5 (5.6) µm) and macroconidia (hyaline, cylindrical, straight and/or slightly curved with one, two or three septa (n = 60) (12.5) 26.5 to 38.5 (44.1) × (4) 5.5 to 7.5 (8.5) µm) were observed. Culture and conidial morphology were in concordance with previous published description of Ilyonectria macrodidyma (Halleen, Schroers & Crous) P. Chaverri & C. Salgado (1,3,4). Identification to species level was confirmed by sequence comparison of four Californian isolates (UCCE958, UCCE959, UCCE960, and UCCE961) with sequences available in GenBank using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA (primers ITS1/ITS4), a portion of the ß-tubulin gene (BT1a/BT1b), and a partial sequence of the mitochondrial small subunit rDNA (NMS1/NMS2) (4). Fungal sequences of isolates from olive from California (GenBank JQ868543 to JQ868554) showed 99 to 100% homology with previously identified and deposited I. macrodidyma isolates in Genbank for all three genes. Pathogenicity of I. macrodidyma in olive cvs. Arbequina, Arbosan, and Koroneiki was investigated using two fungal isolates (UCCE958 and UCCE960) as reported by Petit and Gubler (4). The roots of 10 1-year-old trees per fungal isolate for each olive cultivar were individually inoculated with 25 ml of a 106 conidia/ml spore suspension and placed in a lath house at the UC Davis field station. Additionally, 10 trees per cultivar were inoculated with sterile water as controls. Six months after inoculation, most of the inoculated olive plants showed chlorotic leaves similar to those observed in commercial orchards. Root necrosis for each cv. was expressed as the percentage of root length having lesions (2). No significant difference was observed between isolates and average root necrosis was 29.4, 35.6, and 38.3% in Koroniki, Arbosana, and Arbequina, respectiveley. I. macrodidyma was recovered from symptomatic roots in each of the cvs. and identified based on morphology. No root rot symptoms were observed in the controls. To our knowledge, this is the first report of I. macrodidyma causing root rot of olive trees not only in California but anywhere in the world. References: (1) P. Chaverri et al. Stud. Mycol. 68:57, 2011. (2) M. Giovanetti and B. Mosse. New Phytol. 84:489, 1980. (3) F. Halleen et al. Stud. Mycol. 50:421, 2004. (4) E. Petit and W. D. Gubler. Plant Dis. 89:1051, 2005.

Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses.

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.

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 $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 ß-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 Koch's postulates. Statistical analyses were performed with analysis of variance and Dunnett's 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.

Botryosphaeriaceae Species Spore-Trapping Studies in California Vineyards.

The seasonal abundance of Botryosphaeriaceae spp. spores was studied in California vineyards by using glass microscope slides covered with petroleum jelly placed on grapevine cordons and Burkard volumetric spore traps at seven and two different locations, respectively. Correlation analysis was used to determine which meteorological variables (precipitation, relative humidity, temperature, and wind speed) influenced Botryosphaeriaceae spp. spore release. Among all variables, regression analysis resulted in a strong relationship between spore release and precipitation. Additionally, a positive relationship between irrigation and spore release was also observed in the Riverside County vineyard. During the study period, spore discharge of Botryosphaeriaceae spp. occurred from the first fall rain through the last spring rains, coinciding with September to April. However, based on the results obtained from the spore traps, most spores (over 60%) were trapped following rain events during the winter months of December, January, and February, which coincides with the grapevine pruning season. Botryosphaeriaceae spp. spore release was much lower in fall and early spring (22%) and very few or no spores were trapped in late spring and summer (3%). This work suggests that a delay of pruning time in California may be warranted to reduce grapevine infection because the current timing coincides with the greatest period of spore discharge.

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 ß-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 Tukey's 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.

Neofusicoccum spp. Associated with Stem Canker and Dieback of Blueberry in Chile.

Blueberry (Vaccinium spp.) plantings have significantly increased in Chile during the last decade and, currently, over 10,700 ha are cultivated throughout the country. Among other diseases, stem canker and dieback has been frequently observed in commercial plantations with incidences between 15 and 45%. The aim of this study was to identify and characterize Neofusicoccum spp. causing stem canker and dieback of blueberry in Chile. Three species, N. arbuti, N. australe, and N. parvum, were identified based on colony and conidia morphology, and nucleotide sequence analysis of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2). These Neofusicoccum spp. were found alone or coexisting with Pestalotiopsis spp., Truncatella spp., or Phomopsis spp. Koch's postulates showed all Neofusicoccum spp. isolated from infected plants to be pathogenic when inoculated on blueberry fruit and twigs using both mycelia and conidia suspension. All blueberry cultivars tested, including, Brigitta, Bluecrop, Brightwell, Duke, Elliott, Misty, and O'Neal, were susceptible to Neofusicoccum spp. infection. Pathogenicity tests showed N. parvum to be the most virulent species and Elliott to be the most susceptible cultivar. This report represents the first description of N. arbuti, N. australe, and N. parvum as canker-causing agents on blueberry in Chile.

Pathogenicity of Botryosphaeriaceae Species Isolated from Grapevine Cankers in California.

Fungal species in the family Botryosphaeriaceae have been recently recognized as the most common fungi isolated from grapevine (Vitis vinifera) cankers in California. However, the role of these fungi in causing grapevine dieback as well as their status as canker-causing agents was unknown. Therefore, pathogenicity studies were conducted to determine their importance as grapevine pathogens in California. A total of 72 isolates representing all nine Botryosphaeriaceae species isolated from grapevine cankers from California were used in five different pathogenicity studies. Overall, experiments showed all nine Botryosphaeriaceae species able to infect both young and mature tissues as well as green shoots of the new vegetative growth causing cankers, vascular discoloration, and/or otherwise dark streaking of the wood. However, virulence varied among species. Lasiodiplodia theobromae was the most virulent species followed by Neofusicoccum luteum, N. parvum, and N. australe, all categorized as highly virulent. Botryosphaeria dothidea was considered intermediately virulent and Diplodia mutila, D. seriata, Dothiorella iberica, and D. viticola were shown to be weakly virulent. This study shows species of Botryosphaeriaceae to be much more important pathogens on grapevines than originally thought, and some of them, in view of their virulence, should be considered high risk for causing severe and rapid canker and dieback diseases in the grapevine industry in California.

Identification and Pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the Causal Agents of Bot Canker Disease of Grapevines in Mexico.

Perennial cankers and consequent grapevine dieback are a major problem in vineyards of Sonora and Baja California, the most important grape-production areas of Mexico. In order to identify the canker-causing agents, symptomatic arms, cordons, and trunks were collected from 13 and 6 vineyards in Sonora and Baja California, respectively. Two Botryosphaeriaceae spp., Lasiodiplodia theobromae and Diplodia seriata, were isolated frequently from infected wood and identified based on morphological and cultural characters as well as analyses of nucleotide sequences of three genes, the internal transcribed spacer region (ITS1-5.8S-ITS2), a partial sequence of the ß-tubulin gene, and part of the translation elongation factor 1-α gene (EF1-α). Although both L. theobromae and D. seriata were isolated from grapevine cankers in Baja California, only L. theobromae was found in vines in the Sonora region. Pathogenicity of both species was verified by inoculation of rooted cuttings and green shoots of Thompson Seedless and Chardonnay cultivars. Isolates of L. theobromae were more virulent, based on the extent of spread in the secondary wood and green tissue, than those of D. seriata. These findings confirm L. theobromae and D. seriata as the causal agents of dieback and canker formation of grapevines in northern Mexico.

First Report of Botryosphaeria iberica and B. viticola Associated with Grapevine Decline in California.

Grapevine decline symptoms in California include dead spurs and cordon and trunk dieback due to canker formation in the vascular tissue. Seven Botryosphaeria spp. are known to be associated with grapevine cankers in California, viz. Botryosphaeria australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii (3). Recently, B. iberica and B. viticola also were isolated from grapevine cankers in a field survey that was conducted throughout California. Identification was based on morphological comparisons along with DNA analyses with previously identified isolates from Spain (1,2): B. iberica (CBS115035, ex-type) and B. viticola (CBS117006 and CBS117009, ex-type). DNA sequences of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2), part of the ß-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from B. iberica and B. viticola isolates from California were amplified using primers ITS4/ITS5, Bt2a/Bt2b, and EF-728F/EF-986R, respectively. All DNA sequences of B. iberica and B. viticola from California showed 99 to 100% homology with those previously identified and deposited in GenBank. B. iberica, isolated from grapevine cankers from San Luis Obispo County (central coast), formed colonies on potato dextrose agar (PDA) that were dark green with aerial mycelium, optimum growth at 20 to 25°C, and formed pycnidia after 15 days of incubation at 25°C. Conidia were brown, one-septate, oblong to ovoid with a rounded apex, and measured (20.1-) 22.5 to 23.5 (-27.1) × (8.1) 9.3 to 9.8 (-11.2) µm, length/width ratio = 2.4 (n = 60). B. viticola, isolated from grapevine cankers in Sonoma (north coast), San Luis Obispo, Santa Barbara (south coast), Riverside (southern California), and Yolo (Sacramento Valley) counties, formed colonies on PDA that were dark green to grayish with aerial mycelium, optimum growth at 25°C, and formed pycnidia after 2 weeks. Conidia were brown, one-septate, oval to oblong, and measured (16.6-) 19.3 to 20.3 (-23.5) × (8.1) 9.3 to 9.6 (-11.1) µm, length/width ratio = 2.1 (n = 60). Two isolates of each species were used to complete pathogenicity tests (B. iberica: ATCC MYA-4110, ATCC MYA-4111; B. viticola: ATCC MYA-4115, ATCC MYA-4116). Ten fresh pruning wounds on 15-year-old cv. Zinfandel vines were inoculated per isolate using 50 µl of a 5 × 106 conidia per ml suspension. Twenty control pruning wounds were inoculated with the same amount of sterile water. Twelve months after inoculation, all wood inoculated with B. iberica and B. viticola showed internal necrosis extending 35 to 50 and 30 to 35 mm from the point of inoculation, respectively. Necrosis and extent of vascular discoloration in infected wounds was significantly greater (P < 0.05) than in control inoculations (6.5 mm). B. iberica and B. viticola were reisolated from the necrotic region surrounding all inoculation sites. Representative isolates of B. iberica and B. viticola from California were deposited at the American Type Culture Collection (B. iberica: MYA-4110, MYA-4111; B. viticola: MYA-4112 to MYA-4116). Sequences from the studied DNA regions of all isolates were deposited at GenBank. To our knowledge, this is the first report implicating either species as a cause of grapevine decline in California and B. iberica as a pathogen of Vitis vinifera anywhere in the world. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) A. J. L. Phillips et al. Mycologia 97:513, 2005. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006.

Identification and Distribution of Botryosphaeria spp. Associated with Grapevine Cankers in California.

Botryosphaeria spp. recently have been identified as important grapevine pathogens worldwide. To date, Botryosphaeria rhodina has been the only species associated with cankers on Vitis vinifera in California. A field survey of 166 vineyards in 21 counties was conducted in order to determine the occurrence of other Botryosphaeria spp. in California. In all, 1,735 samples of cankered trunks, cordons, and spurs were collected. Botryosphaeria spp. were the most common fungi isolated from grapevine cankers in California. Morphological identification along with phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of the nuclear ribosomal DNA (rDNA) and a partial sequence of the ß-tubulin gene showed that at least seven Botryosphaeria spp. occur on grapevines in California: B. australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii. Botryosphaeria spp. were found in grapevine cankers in all grape-growing regions surveyed in California, whereas incidence and distribution varied with location. Grapevine cankers in California have been associated mainly with Eutypa dieback. However, the frequent recovery of Botryosphaeria spp. from cankers in this study indicates that the role of these fungi in grapevine health needs to be more carefully considered.