First Report of Diaporthe neoviticola Associated with Wood Cankers of Grapevine in Turkey.

In recent years, delayed bud bursting, cane bleaching, shoot dieback, and cankers in 1-year-old canes and perennial arms were observed in vineyards of the Aegean region (western Turkey). These symptoms were frequently observed on the following major table grape (Vitis vinifera) cultivars: 'Alphonse Lavallée,' 'Cardinal,' 'Sultana Seedless,' and 'Trakya Ilkeren' in 2012. To determine the causal agents, symptomatic woody tissues (0.5 cm2) were sampled from the canes of nine Manisa and four Salihli Cities (13 total) grapevine varieties and were plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). A considerable amount of phomopsis-like fungi were isolated from the symptomatic tissues and fungal colonies were incubated for 2 to 3 weeks to induce sporulation. After incubation for 14 days at 24°C in the dark, white mycelial growth with undulating colony margins, and abundant pycnidia producing hyaline, ellipsoidal, fusoid α-conidia with invisible nuclei, and ß-conidia, were observed on PDA, and they resembled species in the Diaporthaceae (1,2). The α-conidia dimensions were 9.3 to 10.2 × 1.9 to 2.9 µm (avg. 9.7 × 2.4 µm) and ß-conidia were 19 to 24 × 0.5 to 1 µm (avg. 22 × 0.9 µm). For molecular identification, fungal DNA was extracted from mycelial mats and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA, amplified with ITS4 and ITS5 primers) (3) were sequenced and the sequences were compared with those deposited in NCBI GenBank in a BLASTn search. The representative isolate (MBAi43AG) showed 99% homology with Diaporthe neoviticola isolate from New Zealand KC145831.1. The DNA sequence of the identified isolate was submitted to GenBank under accession number KF460427. Pathogenicity tests were conducted under controlled conditions (24°C, 16/8 h day/night, and 70% RH) on 1-cm-diameter, detached green grapevine cv. Cabernet Sauvignon canes (with leaves) using the isolate of D. neoviticola specified above. The shoots were wounded by creating a 5-mm-diameter incision with a sterile scalpel. An agar disc with mycelia and pycniospores was placed into each wound and covered with Parafilm. Sterile PDA plugs were used as mock inoculum for the control plants. There were 10 replicates per treatment and the experiment was repeated twice. After 1 month of incubation, the green shoots were examined for the extent of superficial blackish lesions. The average lesion length on inoculated shoots was 18.2 mm for D. neoviticola. No lesions were observed in the control shoots. The fungal isolate was successfully re-isolated from 96% of inoculated shoots to fulfill Koch's postulates. To our knowledge, this is the first report of D. neoviticola causing wood canker and dieback of shoots on grapevine in Turkey. References: (1) R. R. Gomes. Persoonia 31:1, 2013. (2) D. Udayanga et al. Fungal Diversity 56:157, 2012. (3) J. M. van Niekerk et al. Australas. Plant Pathol. 34:27, 2005.

First Report of Wood Canker Caused by Botryosphaeria dothidea, Diplodia seriata, Neofusicoccum parvum, and Lasiodiplodia theobromae on Grapevine in Turkey.

The Aegean region (western Turkey) is the center of table, raisin, and wine grape cultivation. During the 2012 growing season, wood canker symptoms were observed in vineyards in Manisa city. Symptoms adjacent to pruning wounds, including shoot dieback and wedge-shaped wood discolorations observed in cross section, were among the most prevalent symptoms of the vines. To identify the causal agents, symptomatic woody tissues were surface disinfested with 95% ethanol and flame-sterilized and the discolored outer bark was cut away. The internal tissues (0.5 cm2) were excised from cankers of vines and plated onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed of growth, and colony color, resembled species in the Botryosphaeriaceae family. According to morphological characteristics, four different groups have been identified based on visual discrimination. After DNA extraction, ribosomal DNA fragments (ITS1-5.8S-ITS2) (2) amplified with ITS4 and ITS5 primers were sequenced and sequences were compared with those deposited in NCBI GenBank database. Four different Botryosphaeriaceae isolates were identified, including Botryosphaeria dothidea (MBAi25AG), Diplodia seriata (MBAi23AG), Lasiodiplodia theobromae (MBAi28AG), and Neofusicoccum parvum (MBAi27AG) (Accession Nos. KF182329, KF182328, KF182331, and KF182330, respectively) with species nomenclature based on Crous et al. (1). Pathogenicity tests were conducted under greenhouse conditions (24°C, 16/8-h day/night, 70% RH) on 1-year-old own rooted grapevine (Vitis vinifera) cv. Sultana Seedless seedlings using one isolate from each of the Botryosphaeriaceae species specified above. Stems of grapevine seedlings were wounded by removing bark with 4-mm cork borer and fresh mycelial plugs were inoculated into the holes and covered with Parafilm. Sterile PDA plugs were placed into the wounds of control seedlings. Five vines were inoculated per isolate. The experiment was repeated twice. After 4 months of incubation, grapevine seedlings were examined for the extent of vascular discoloration and recovery of fungal isolates. Mean lesion lengths on wood tissues were 85.3, 17.2, 13.9, and 13.1 mm for N. parvum, B. dothidea, L. theobromae, and D. seriata, and 6.3 mm for control. Each fungal isolate was successfully re-isolated from inoculated seedlings to fulfill Koch's postulates. To our knowledge, this is the first report of multiple species in the Botryosphaeriaceae causing wood canker and dieback on grapevine in Turkey. These results are significant because Botryosphaeriaceae species are known causal agents of grapevine trunk disease worldwide (3). References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) J. R. Urbez-Torres. Phytopathol. Mediterr. 50:S5, 2011.

First Report of Geosmithia pallida Causing Foamy Bark Canker, a New Disease on Coast Live Oak (Quercus agrifolia), in Association with Pseudopityophthorus pubipennis in California.

Declining coast live oak (Quercus agrifolia) trees have been observed since 2012 throughout urban landscapes in Los Angeles, Orange, Riverside, Santa Barbara, Ventura, and Monterey counties in California. Symptoms causing branch dieback and tree death included a cinnamon-colored gum seeping through multiple 0.95-mm-diameter entry holes on the bole, followed by a prolific, cream-colored foamy liquid. Beneath the outer bark was phloem and xylem necrosis. Fifty 1- to 2.5-mm adult and larval beetles were collected. Adults fit the morphological description of Pseudopityophthorus pubipennis (western oak bark beetle) (R. Rabaglia, personal communication), and ~800 bp of the mitochondrial COI gene was amplified for three beetles using primer pairs and methods previously described (2,3). All three sequences were identical (GenBank Accession Nos. KJ831289 to 91) and a BLAST search confirmed the closest match (94%) as P. pubipennis. Necrotic wood tissues collected from two trees in each county were cultured on potato dextrose agar amended with 0.01% tetracycline (PDA-tet), and incubated at 25°C for 1 week. Ochre-colored cultures with plane or radially furrowed velutinous mycelium were consistently produced. Fifty conidia each measured from two isolates were 3.66 ± 0.04 µm × 1.77 ± 0.03 µm, and arranged in non-persistent conidial chains, at first roughly parallel, becoming tangled with age. These fungal colonies were observed within gallery walls. The rDNA internal transcribed spacer (ITS) was amplified using primer pairs and methods previously described (5). Three isolates were sequenced and matched 100% to known sequences of Geosmithia pallida in GenBank; sequences of two isolates (UCR2208 and UCR2210) were deposited in GenBank (KJ468687 and KJ468688). Pathogenicity tests were performed by inoculating twelve 27.0-cm detached coast live oak shoots for each isolate with a spore suspension of G. pallida (UCR2208 and UCR2210) and sterile distilled water for controls. A 2-mm-wide, 3-mm-deep hole was drilled into the center of each shoot, 20 µl of a 106 conidia/ml spore suspension was pipetted into the hole, and sealed with Vaseline and Parafilm. The experiment was repeated twice. After 4 weeks in a moist chamber at 25°C, lesions produced by G. pallida averaged 8.3 cm and was significantly longer (ANOVA; P < 0.0001) from the control (average 0.4 cm). G. pallida was re-isolated from all inoculated plants and identified by colony morphology. P. pubipennis is a native beetle, common as a secondary agent, and previously not associated with disease. However, cryptic species may be common among bark and ambrosia beetles (4). A larger sample (i.e., populations and loci) is needed to determine the precise taxonomic status of P. pubipennis. G. pallida was shown to inhibit root growth of Q. petraea by 25% in Europe (1), appears to have affinities with a range of subcorticolous insects, and is widely distributed (5), but there is no published record of the fungus occurring in the United States. This is the first report of G. pallida causing foamy bark canker in association with P. pubipennis on Q. agrifolia in California. Results suggest this new disease complex is causing decline of Q. agrifolia throughout the state. References: (1) D. Cizková et al. Folia Microbiol. 50:59, 2005. (2) A. I. Cognato and F. A. H. Sperling. Mol. Phylogenet. Evol. 14:445, 2000. (3) A. I. Cognato et al. Mol. Phylogenet. Evol. 36:494, 2006. (4) B. H. Jordal and M. Kambestad. Mol. Ecol. Res. 14:7, 2014. (5) M. Kolarík et al. Mycol. Res. 108:1053, 2004.

Identification of Species of Botryosphaeriaceae Causing Bot Gummosis in Citrus in California.

Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as 'Eureka' lemon, 'Valencia', 'Washington Navel', 'Fukumoto', grapefruit, 'Satsuma', and 'Meyer' lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, ß-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.

Fusarium euwallaceae sp. nov.--a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California.

The invasive Asian ambrosia beetle Euwallacea sp. (Coleoptera, Scolytinae, Xyleborini) and a novel Fusarium sp. that it farms in its galleries as a source of nutrition causes serious damage to more than 20 species of live trees and pose a serious threat to avocado production (Persea americana) in Israel and California. Adult female beetles are equipped with mandibular mycangia in which its fungal symbiont is transported within and from the natal galleries. Damage caused to the xylem is associated with disease symptoms that include sugar or gum exudates, dieback, wilt and ultimately host tree mortality. In 2012 the beetle was recorded on more than 200 and 20 different urban landscape species in southern California and Israel respectively. Euwallacea sp. and its symbiont are closely related to the tea shot-hole borer (E. fornicatus) and its obligate symbiont, F. ambrosium occurring in Sri Lanka and India. To distinguish these beetles, hereafter the unnamed xyleborine in Israel and California will be referred to as Euwallacea sp. IS/CA. Both fusaria exhibit distinctive ecologies and produce clavate macroconidia, which we think might represent an adaption to the species-specific beetle partner. Both fusaria comprise a genealogically exclusive lineage within Clade 3 of the Fusarium solani species complex (FSSC) that can be differentiated with arbitrarily primed PCR. Currently these fusaria can be distinguished only phenotypically by the abundant production of blue to brownish macroconidia in the symbiont of Euwallacea sp. IS/CA and their rarity or absence in F. ambrosium. We speculate that obligate symbiosis of Euwallacea and Fusarium, might have driven ecological speciation in these mutualists. Thus, the purpose of this paper is to describe and illustrate the novel, economically destructive avocado pathogen as Fusarium euwallaceae sp. nov. S. Freeman et al.

First Report of Togninia minima Perithecia on Esca- and Petri-Diseased Grapevines in South Africa.

Esca and petri diseases are important grapevine trunk diseases in South Africa and most other grape-producing countries. The causal pathogens are Phaeomoniella chlamydospora and several species of Phaeoacremonium. In total, 25 species of Phaeoacremonium have been isolated from grapevines of which seven species have been linked to Togninia teleomorphs obtained through in vitro mating studies (3). Of these species, only perithecia of T. minima, T. fraxinopennsylvanica, and T. viticola have been found on grapevines in California (1,2,4). T. minima is heterothallic, and although both mating types are present in South African vineyards, perithecia have never been observed (3). In the current study, grapevine cordons and trunks were collected from vineyards and rootstock mother vines within Western Cape Province for examination in the laboratory under a dissecting microscope. The grapevines displayed general decline symptoms, including reduced vegetative growth, dead or dying shoots and cordons, as well as internal vascular streaking and/or a red/black/brown margin next to decayed wood typically associated with esca and petri disease. Rootstock mother vines were apparently healthy, although many old, cracked pruning wounds were visible. Togninia-like perithecia with distinctive long necks were found along the wood crevices, often on old pruning wounds. The perithecia were removed and placed on microscope slides with sterile water. Structures were measured and slides were washed with 500 µl of sterile water onto potato dextrose agar amended with chloramphenicol (250 mg/liter). Ascospores were allowed to germinate overnight to obtain single ascospore colonies. Perithecia were found on cultivars Muscat d' Alexandrie and Pinotage (Vitis vinifera) at Stellenbosch in May 2011 and on Ramsey (V. champinii) rootstock mother vines at Slanghoek in June 2012. Perithecia were globose to subglobose, black, and often embedded in the wood tissue but also present on the surface of the wood. The length of the necks was 250 to 300 × 47.5 to 55 µm. The asci were hyaline and ranged from 16 to 25 × 3.5 to 5 µm. Ascospores were hyaline, ellipsoid, and ranged from 5 to 6 × 1.5 to 2 µm. These measurements were similar to those reported by Mostert et al. (3) and Rooney et al. (4). Colony growth was typical of T. minima. DNA was extracted from the colonies and the partial betatubulin gene was amplified and sequenced using the primers T1 and Bt2b. Sequences were deposited into GenBank (JX962864 to 67). Based on a megablast search of the NCBI's GenBank nucleotide database, 100% similarity was found with other T. minima sequences (JQ691670.1, HQ605018.1, HQ605014.1; identities = 647/647 [100%], gaps = 0/647 [0%]). To our knowledge, this is the first report on the occurrence of T. minima perithecia on grapevines in Western Cape Province of South Africa. The removal of dead spurs and cordons will be instrumental in lowering the inoculum originating from perithecia, especially in rootstock mother blocks where no control strategies are applied for petri disease or esca. Spore trapping studies are currently in progress to study spore release patterns in order to determine whether pruning wounds are at risk during traditional pruning periods. References: (1) A. Eskalen et al. Plant Dis. 89:528, 2005. (2) A. Eskalen et al. Plant Dis. 89:686, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006. (4) S. Rooney-Latham et al. Plant Dis. 89:867, 2005.

First Report of Wood Canker Caused by Neoscytalidium dimidiatum on Grapevine in California.

In May 2012 in the Coachella valley, Riverside County, California, the decline of vines in table grape (Vitis vinifera) vineyards was observed. Foliar symptoms consisted of shoot blight with wilting and necrosis of leaves and drying and shriveling of berries. In some cases, the entire vine collapsed in the middle of the growing season (apoplexia). Wood cankers in the spurs, cordons, and trunks of affected vines were also present. The nine isolates recovered from the cankers were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers based on morphological characteristics and DNA sequence comparisons. Two isolates were grown on potato dextrose agar (PDA) medium and a total of 50 conidia were measured per isolate. Conidia were ellipsoid to ovoid, with a truncate base and an acutely rounded apex, initially aseptate, becoming brown and two-celled at maturity, 7.2 ± 1.2 µm × 3.8 ± 0.4 µm. The rDNA internal transcribed spacer (ITS), and ß-tubulin (BT) loci were amplified using primer pairs and methods previously described (4). A total of five isolates were sequenced. The DNA sequences of one N. dimidiatum grapevine isolate (UCR-Neo1) were deposited in the GenBank database (ITS, KC937066; BT, KC937067). Pathogenicity tests were performed by inoculating 12 grape cuttings cv. Thompson Seedless with isolate UCR-Neo1 and 12 control cuttings with sterile medium using a technique previously described (1). The experiment was repeated twice. After 20 weeks of incubation period in the greenhouse, the lesions length produced by N. dimidiatum averaged 13.5 mm and was significantly longer (P < 0.05) from the control (average 3 mm). N. dimidiatum was reisolated from all the inoculated plants and identified by colony morphology. The incidence of N. dimidiatum in table grape vineyards of the Coachella valley has been estimated at 15%, with nine vines infected out of 60 vines total. This pathogen has been identified in California in walnut nursery causing the death of trees due to the development of canker at the graft union (2). N. dimidiatum has also been identified as the causal agent of shoot blight, canker, and gummosis on citrus in Italy (3). The crop is also being grown in the Coachella valley and these findings warrant further investigation in order to determine the host range, distribution, and incidence of this pathogen in the area. References: (1) K. Baumgartner et al. Plant Dis. 97:912, 2013. (2) S. F. Chen et al. Plant Dis. 97:993, 2013. (3) G. Polizzi et al. Plant Dis 93:1215, 2009. (4) J. R. Urbez-Torres et al. Plant Dis. 92:519, 2008.

Identification and Pathogenicity of Fungal Pathogens Associated with Stem-End Rot of Avocado in California.

Stem-end rot of harvested avocado fruit commonly occurs wherever the crop is cultivated. Multiple fungal species have been described as causal agents. To determine the causal pathogens of stem-end rot in California, fungal isolations were conducted from symptomatic fruit, and fungi were identified by morphological and molecular techniques. In 2010 and 2011, a total of 177 isolates were recovered from 290 avocado fruit collected from seven orchards in one of the major avocado growing areas in Southern California. The majority of isolates was identified as Neofusicoccum luteum (65%), with the remainder either as Colletotrichum gloeosporioides (33%) or Phomopsis sp. (2%). In a pathogenicity test, N. luteum caused significantly (P < 0.05) more severe stem-end rot than either C. gloeosporioides or Phomopsis sp. No significant (P > 0.05) differences in stem-end rot severity were observed between inoculations with N. luteum isolated from fruit stem-end rot and N. luteum or N. parvum isolated from branch cankers. This confirms that stem-end rot of avocado can be initiated by fungi causing branch cankers. Although low humidity and rainfall during much of the growing and harvest seasons in California are considered unfavorable conditions for the development of avocado stem-end rot, the identification of the causal pathogens is of value when decays have to be managed during outbreaks, and it stresses the importance of managing branch cankers.

Effect of Fungicide Application on the Management of Avocado Branch Canker (Formerly Dothiorella Canker) in California.

Members of the Botryosphaeriaceae family have been associated with branch cankers of avocado trees (Persea americana) in California. Canker infections are initiated by spores entering the host plant through fresh wounds such as pruning wounds. With high-density planting becoming more common in the California avocado industry, more intensive pruning may increase the occurrence of branch canker. The objective of this study was to evaluate the preventive ability of some commercial fungicides belonging to different chemical families against fungal pathogens associated with avocado branch canker. Initially, 12 fungicides were tested in vitro for their effect on the inhibition of mycelial growth of three isolates of Dothiorella iberica and isolates (five per species) of Neofusicoccum australe, N. luteum, N. parvum, and Phomopsis sp. Subsequently, azoxystrobin, fludioxonil, metconazole, and pyraclostrobin, selected because of their low effective concentrations that reduce 50% of mycelial growth (EC50 values), and myclobutanil, selected for its high EC50 value, were tested in two field experiments. Azoxystrobin and fludioxonil were used in a premix with propiconazole and cyprodinil, respectively, in field trials. Significant differences (P < 0.05) were observed among fungicides in field trials. Azoxystrobin + propiconazole had the highest percent inhibition at 52 and 62% (internal lesion length) in trial 1 and trial 2, respectively, although this level of inhibition was not significantly different from that of metconazole. A significant correlation (r = 0.51, P < 0.05) was observed between internal lesion length data in the field experiment and EC50 data from in vitro fungicide screening. Application of azoxystrobin + propiconazole and metconazole can play a key role in protecting Californian avocado against fungi causing avocado branch canker.

First Report of Multiple Species of the Botryosphaeriaceae Causing Bot Canker Disease of Indian Laurel-Leaf Fig in California.

Indian laurel-leaf fig (Ficus microcarpa L.) is a commonly used indoor and outdoor ornamental tree. F. microcarpa is most frequently encountered as lining city streets, especially in warmer southern California climates. A disease known as 'Sooty Canker,' caused by the fungus Nattrassia mangiferae (Syd. & P. Syd) B. Sutton & Dyko, is particularly devastating on F. microcarpa. Disease symptoms are characterized by branch dieback, crown thinning, and if the disease progresses to the trunk, eventual tree death (2). Recent taxonomic revisions have renamed Nattrassia mangiferae as Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A. J. L. Phillips (1). An initial survey conducted during the spring of 2011 across four cities in Los Angeles County included, Culver City, Lakewood, Santa Monica, and Whittier. Five symptomatic branches per city were collected from trees showing branch cankers and dieback. Pieces of symptomatic tissue (2 mm2) were plated onto one-half-strength potato dextrose agar. Most isolates initially identified by morphological characteristics, such as growth pattern, speed of growth, and colony color, resembled those in the Botryosphaeriaceae (4). Two representative isolates from each site location were sequenced. Sequences obtained from amplification of the internal transcribed spacer region (ITS1-5.8rDNA-ITS2) and the ß-tubulin gene were compared in a BLAST search in GenBank. Results identified isolates as Botryosphaeria dothidea (identity of 99% to EF638767 and 100% to JN183856.1 for ITS and ß-tubulin, respectively); Neofusicoccum luteum (100% to EU650669 and 100% to HQ392752); N. mediterraneum (100% to HM443605 and 99% to GU251836); and N. parvum (100% to GU188010 and 100% to HQ392766) and have been deposited in GenBank with the following accession numbers: JN543668 to JN543671 (ITS) and JQ080549 to JQ080552 (ß-tubulin). Pathogenicity tests were conducted in the greenhouse on 6-month-old F. microcarpa with one isolate from each previously listed fungal species. Five plants per isolate were stem-wound inoculated with mycelial plugs and wrapped with Parafilm. Uncolonized agar plugs were used as a control. Inoculations were later repeated a second time in the same manner for a total of 10 plants per isolate. Plants were observed for 6 weeks and destructively sampled to measure vascular lesion lengths. Mean vascular lesion lengths were 26, 22, 54, and 46 mm for B. dothidea, N. luteum, N. mediterraneum, and N. parvum, respectively. The mean lesion lengths for all isolates were significantly different (P = 0.05) from the control. Each species was consistently recovered from inoculated plants, except the control, thus fulfilling Koch's postulates. To our knowledge, this is the first report on the pathogenicity of multiple Botryosphaeriaceae species causing branch canker and dieback on F. microcarpa in California. These results are significant since trees along sidewalks in southern California are often crowded and undergo extensive root and branch pruning and some Botryosphaeriaceae spp. are known to enter its host through wounds caused by pruning or mechanical injury (2,3). Further sampling is imperative to better assess the distribution of these canker-causing fungal pathogens on F. microcarpa. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) D. R. Hodel et al. West. Arborist 35:28, 2009. (3) V. McDonald et al. Plant Dis. 93:967, 2009. (4) B. Slippers et al. Fungal Biol. Rev. 21:90, 2007.

First Report of a Fusarium sp. and Its Vector Tea Shot Hole Borer (Euwallacea fornicatus) Causing Fusarium Dieback on Avocado in California.

Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 µm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 µm, 9.2 to 17.2 × 3.4 to 4.8 µm, and 13.5 to 17.6 × 4.3 to 4.7 µm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Koch's postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California. References: (1) W. Danthanarayana. Tea Quarterly 39:61, 1968. (2) H. Kajimura and N. Hijii. Ecol. Res. 7:107, 1992; (3) Mendel et al., Phytoparasitica, DOI 10.1007/s12600-012-0223-7, 2012. (4) R. J. Rabaglia. Annals Entomol. Soc. Amer. 99:1034, 2006.

First Report of Spencermartinsia viticola, Neofusicoccum australe, and N. parvum Causing Branch Canker of Citrus in California.

Dothiorella gummosis and canker on citrus is generally viewed as a minor disease but can result in serious decline of trees. Symptoms, mostly found on branches, include grayish-to-brown cast on cankered bark, which can extend into the xylem. Dothiorella gummosis was earlier believed to be caused by Dothiorella gregaria (2). In a continuing survey on citrus in six California counties (Fresno, Riverside, San Diego, San Luis Obispo, Tulare, and Ventura) in 2010, branch cankers were collected. Small pieces of symptomatic tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. Fungi most frequently isolated were initially identified as Botryosphaeriaceae based on morphological characters (1,3). Total genomic DNA was PCR amplified with primers Bt2a/2b for the ß-tubulin (BT); EF1-728F/986R for the elongation factor α-1 (EF); and ITS4/5 for the internal transcribed spacer ITS1-5.8S-ITS2 regions (3). Sequences were compared in a BLAST search. Spencermartinsia viticola UCP105 was isolated from cv. Parent Washington on Sour Orange rootstock in Tulare County, Neofusicoccum australe UCR1110 from cv. Satsuma in Riverside County, and N. parvum UCR1166 from cv. Meyer Lemon on Volkameriana rootstock in Ventura County. Sequences of UCP105, UCR1110, and UCR1166 have been deposited in GenBank under Accession Nos. JF271766, JF271776, and JF271780 for BT; JF271784, JF271793, and JF271796 for EF; and JF271748, JF271758, and JF271762 for the ITS regions. The sequences matched with isolates in GenBank as follows: ITS region of strain UCP105-98% match with Accession Nos. AY905556-8; BT of strain UCR1110-99% with GU251879-80; and EF of strain UCR1166-98% with GU251238. Pathogenicity tests were conducted by inoculating green shoots of healthy citrus trees similar to cultivar/rootstock from which each isolate was obtained. Fresh wounds were made on 1-year-old citrus shoots with a 3-mm cork borer, and the freshly wounded surfaces were inoculated with 3-mm mycelial plugs from 5-day-old cultures on PDA-tet. Control shoots were inoculated with sterile agar plugs and each treatment had 10 replicates. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm to prevent desiccation. Shoots were incubated at 25°C in moist chambers for 4 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 6.4, 7.0, and 6.9 cm for UCP105, UCR1110, and UCR1166, respectively, which were significantly (P = 0.05) different from the control (0.8 cm). The three isolates were reisolated from symptomatic tissues of inoculated shoots to confirm their pathogenicity. This test was repeated and similar results were obtained. Results indicate that there are multiple species in the Botryosphaeriaceae family causing symptoms on citrus that were previously believed to be caused by D. gregaria. To our knowledge, this is the first report of S. viticola, N. australe, and N. parvum on citrus in California. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) V. McDonald et al. Plant Dis. 93:967, 2009. (3) B. Slippers et al. Mycologia 96:83, 2004.

First Report of Raffaelea canadensis Causing Laurel Wilt Disease Symptoms on Avocado in California.

Laurel wilt disease is a newly described vascular disease of redbay (Persea borbonia (L.) Spreng.) and other members of the Lauraceae family in the southeastern United States. The disease, caused by the fungus Raffaelea lauricola and vectored by a nonnative redbay ambrosia beetle (Xyleborus glabratus Eichhoff), was first detected in Georgia in 2003 (1). Laurel wilt has caused extensive mortality of native redbay in Georgia, Florida, South Carolina, and recently, Mississippi. The avocado, Persea americana, is in the Lauraceae family and has been shown to be susceptible to the laurel wilt pathogen in Florida (3). The potential spread of this pathogen into California is of concern to the commercial avocado industry. During a survey in 2010 in a Temecula, CA avocado orchard with a history of root rot, an avocado (cv. Hass) tree with a diameter at breast height (DBH) of 45 cm was found to be showing typical laurel wilt disease symptoms. The crown was approximately 80% declined and exhibited dead branches without leaves. Black-to-brown discolored sapwood under the bark and many ambrosia beetle exit holes within 1 to 1.5 m up the bole were also observed. A Raffaelea sp. was consistently isolated from symptomatic branch tissue (from two different branches) plated onto cycloheximide-streptomycin malt agar (2) and incubated at room temperature for 2 weeks. Small subunit (18S) sequences of rDNA (approximately 1,150 bp) of three Raffaelea isolates were amplified using primers NS1 and NS4 (4) and deposited into GenBank under Accession Nos. JF327799, JF327800, and JF327801. A BLASTn search of all three sequences revealed high homology (98, 99, and 98% respectively) to an accession of R. canadensis associated with a species of ambrosia beetle (GenBank Accession No. AY858665). Pathogenicity testing was conducted by pipetting 50 µl of a 105 conidia per ml suspension of each of two isolates (UCR1080 and UCR1081) into five 2-mm-diameter holes on each of two avocado (cv. Hass) trees (10 to 15 cm DBH). Isolate UCR1080 was inoculated into three holes on Tree 1 and two holes on Tree 2. Isolate UCR1081 was inoculated into two holes on Tree 1 and three holes on Tree 2. Sterile water was used as a control in five 2-mm-diameter holes on each tree. Holes were drilled to the cambium within 1 to 2 m up the bole using a 0.157-cm electric drill. Four months later, phloem tissue was peeled back, lesion lengths were measured, and pieces of necrotic tissue were cultured for completion of Koch's postulates. R. canadensis was consistently reisolated from necrotic tissue but not from control treatments. To our knowledge, this is the first report of R. canadensis associated with wilt on avocado in California. R. canadensis is closely related to R. lauricola, however, its impact on the California avocado industry is unknown at this time. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 111:337, 2010. (3) A. E. Mayfield et al. Plant Dis. 92:976, 2008. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

First Report of Eutypella spp. Associated with Branch Canker of Citrus in California.

Eutypella is one of the few genera in the Diatrypaceae considered plant pathogens (1). In California, E. vitis and other members of the Diatrypaceae cause branch and trunk canker on grapevine (3,4). Eutypella spp. have not previously been documented as pathogens of citrus. In a 2010 survey on citrus branch canker and dieback in six citrus-growing counties of California, four isolates of Eutypella species were detected in Riverside and San Diego counties. Canker symptoms included dieback and bark cracking, and cuts made through symptomatic trees showed that the cankers were expanding through the center of the tree. Branch samples were collected from 10 trees per orchard and 5 to 10 orchards per county (102 trees for two counties). Pieces of symptomatic tissue (1 to 2 mm2) were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated at 25°C for 4 days. All isolates were identified by morphological and molecular characteristics. PCR of isolates was performed in a thermal cycler using two primer pairs, ITS4/5 and Bt2a/2b for amplifying the internal transcribed spacer (ITS1), 5.8S, and ITS2 region and the ß-tubulin gene, respectively (2,3). PCR products were sequenced at the University of California, Riverside Genomics Core and the sequences compared in a BLAST search. Four isolates identified as Eutypella spp. included two (UCR1088 and UCR1101) from San Diego County and two (UCR1148 and UCR1149) from the Riverside County samples. The sequences were deposited in GenBank (HQ880579, JF758610, HQ880581, and HQ880582 and HQ880583, JF758611, HQ880585, and HQ880586 for the ITS regions and ß-tubulin gene, respectively. ITS sequences for UCR1088 and UCR1101 had 98 and 100% match, respectively, to Eutypella spp. ITS sequences in GenBank (GQ293959 to GQ293961), while UCR1148 and UCR1149 matched 99% (GQ293956 to GQ293958). On the basis of morphological characteristics, UCR1088 and UCR1101 were similar to Eutypella spp. group 1, while UCR1148 and UCR1149 were similar to Eutypella spp. group 3 (4). Pathogenicity tests were conducted with all four isolates on detached shoots from healthy citrus trees of the same cultivar/rootstock from which each isolate was obtained. One wound per shoot was made on 1-year-old, green, detached shoots using a 3-mm-diameter cork borer and the wounded surfaces were inoculated with 3-mm-diameter mycelial plugs of 5-day-old cultures of each isolate growing on PDA-tet. Inoculated wounds and shoot ends were covered with petroleum jelly and wrapped with Parafilm (3). Control shoots were inoculated with sterile agar plugs. There were 10 inoculated shoots per isolate and noninoculated control treatment. Shoots were incubated at 25°C in moist chambers for 6 weeks. Lesions similar to those on the original infected shoots were observed on all inoculated shoots except the control treatment. Reisolation and identification of fungi from inoculated and control shoots were done using methods described above. Inoculated isolates were recovered from 100% of inoculated shoots but none was recovered from noninoculated shoots, indicating association of Eutypella spp. with citrus branch canker. To our knowledge, this is the first report of Eutypella spp. associated with cankers on citrus in California. References: (1) B. Piskur et al. Plant Dis. 91:1579, 2007. (2) B. Slippers et al. Mycologia 96:83, 2004. (3) F. P. Trouillas and W. D. Gubler. Plant Dis. 94:867, 2010. (4) F. P. Trouillas et al. Mycologia 102:319, 2010.

First Report of Bot Canker Caused by Diplodia corticola on Coast Live Oak (Quercus agrifolia) in California.

Sharp decline and mortality of coast live oak (Quercus agrifolia) has been observed in San Diego County, CA since 2002. Much of this decline has been attributed to a new pest in California, the goldspotted oak borer (GSOB, Agrilus coxalis) (1). Symptoms include crown thinning, bark cracking and/or peeling, patches of stain (1 to 10 cm in diameter), bleeding on the bole, and tree death and are most often observed on trees with a diameter at breast height (DBH) >30 cm. In 2008, a Botryosphaeria sp. was recovered from necrotic tissue of bleeding bole cankers from GSOB-affected trees in Jamul, CA. Zone lines separated dead and live tissue in affected phloem and xylem. Pycnidia were observed on the bark surface of the infected host. Fifty conidia averaging 32 × 18 µm, one-septate with age, and morphologically similar to conidia described by Úrbez-Torres et al. were observed (4). Oak stands with tree mortality were surveyed in GSOB-infested and -uninfested sites over eight locations throughout San Diego and Riverside counties in 2009 and 2010. Symptomatic tissue or conidia from pycnidia of affected trees, plated onto potato dextrose agar amended with 0.01% tetracycline and incubated at 25°C for 1 week, consistently produced cultures with dense, wooly, olive-green mycelium. Mycelia fit the description of Botryosphaeria corticola A.J.L. Phillips, Alves et Luque (anamorph Diplodia corticola) (2). The resulting amplified ITS4/5 region of two sequences matched 100% to published D. corticola sequences (GU799472 and GU799460) (4). These sequences were deposited with NCBI GenBank (HM104176 and HM104177). Koch's postulates were conducted by inoculating 2-mm-diameter holes on five coast live oak trees with D. corticola. Holes were drilled to the cambium at 2 to 4 locations per tree within 1 to 2 m up the bole using a 0.157-cm portable electric drill. Trees ranged from 3.7- to 32.4-cm DBH. Either single agar plugs from two isolates each of a 7-day-old culture (UCR454 and UCR793) or noncolonized agar plugs as uninoculated controls were inserted into the holes and then covered with petroleum jelly and Parafilm. Average temperature was 10°C, relative humidity of 64%, and no precipitation during inoculation. Inoculations were conducted at a location in San Diego County uninfested by GSOB and repeated twice. After 3.5 months, bark was removed from inoculation sites. Average lesion length was not significantly different between inoculations, thus data were combined (one way analysis of variance [ANOVA]; P = 0.05). Lesions averaged 13.9 × 2.3 cm and were significantly different (n = 30; one way ANOVA; P = 0.05) from controls that measured 0.31 × 0.3 cm. Staining was observed around the inoculation points on all trees and three trees exhibited bleeding. Necrotic tissue was observed in the phloem and 3 mm into the xylem tissue, where the lesion had extended up and down the grain. D. corticola was consistently reisolated from necrotic tissue but not from control treatments. B. corticola was originally described as a canker pathogen on Quercus spp. in the western Mediterranean (2), and is known to contribute to the decline of cork oak (Q. suber) in the region (3). To our knowledge, this is the first report of D. corticola causing bot canker on coast live oak in California. References: (1) T. W. Coleman and S. J. Seybold. U. S. For. Serv. R5-PR-08, 2008. (2) A. Correia et al. Mycologia 96:598, 2004. (3) J. Luque et al. For. Pathol. 38:147, 2008. (4) J. R. Úrbez-Torres et al. Plant Dis. 94:785, 2010.

First Report of Neofusicoccum australe, N. luteum, and N. parvum Associated With Avocado Branch Canker in California.

In 1953, branch cankers on California avocado (Persea americana Mill.) trees were attributed to a Botryosphaeria anamorph, Dothiorella gregaria (teleomorph B. ribis) (2), and the disease was known as Dothiorella canker. Since this time, it has been suggested that this fungus should probably be classified as Fusicoccum aesculi Corda (teleomorph B. dothidea) (3). To our knowledge, B. dothidea is the only reported Botryosphaeriaceae species causing Dothiorella canker on avocado in California. Between the summer of 2008 and the winter of 2009, five trees from each of eight avocado orchards in five counties (San Diego, Riverside, Ventura, Santa Barbara, and San Luis Obispo) were surveyed for Dothiorella canker symptoms to verify the associated Botryosphaeriaceae species. Typical Dothiorella canker symptoms observed included darkened and friable bark with a dried, white, powdery exudate. Underneath the bark, cankers were variable in shape and some penetrated into the heartwood. Small sections of tissue (0.5 cm2) were excised from two to four separate cankers per tree and placed onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed, and colony color, were in the Botryosphaeriaceae with the following percent recovery by county: Riverside-40 and 100% (site 1 and 2, respectively); San Diego-60% (site 3); Ventura-42 and 53% (site 4 and 5, respectively); Santa Barbara-33% (site 6); and San Luis Obispo-32 and 60% (site 7 and 8, respectively). Pycnidia of Botryosphaeriaceae species were also observed on old diseased avocado tree branches. Sequenced rDNA fragments (ITS1, 5.8S rDNA, ITS2, amplified with ITS4 and ITS5 primers) were compared with sequences deposited in GenBank. Four different Botryosphaeriaceae species were identified and included Neofusicoccum australe, B. dothidea, N. luteum, and N. parvum, with species nomenclature based on the work of Crous et al. (1). Pathogenicity tests were conducted in the greenhouse on 1-year-old avocado seedlings, cv. Hass, with one randomly chosen isolate from each of the Botryosphaeriaceae species noted above. Four replicate seedlings were stem-wound inoculated with a mycelial plug and covered with Parafilm. Sterile PDA plugs were applied to four seedlings as a control. Over a period of 3 to 6 months, seedlings were assessed for disease symptoms that included browning of leaf edges and shoot dieback. Mean vascular lesion lengths on stems were 64, 66, 64, and 18 mm for B. dothidea, N. parvum, N. luteum, and N. australe, respectively. Each fungal isolate was consistently reisolated from inoculated seedlings, thus fulfilling Koch's postulates. To our knowledge, this is the first report of N. australe, N. luteum, and N. parvum recovered from branch cankers on avocado in California. These results are significant because Botryosphaeriaceae canker pathogens are known to enter the host plant through fresh wounds (pruning, frost, and mechanical). With high-density planting becoming more common, which requires intensive pruning, the transmission rate of these pathogens could increase in California avocado groves. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) F. F. Halma and G. A. Zentmyer. Calif. Avocado Soc. Yearb. 38:156, 1953. (3) W. F. T. Hartill and K. R. Everett. N. Z. J. Crop Hortic. Sci. 30:249, 2002.

First Report of Aspergillus carbonarius Causing Sour Rot of Table Grapes (Vitis vinifera) in California.

During the 2006 growing season, grape berries in several Red Globe vineyards in Kern County, California were found exhibiting black fungal sporulation and typical sour rot symptoms. Symptoms included berry cracking and leakage along with a pungent vinegar smell. In California, sour rot (also known as summer bunch rot) has been attributed to a complex of microorganisms that invade ripe berries following injury. Fungi typically associated with sour rot include Aspergillus niger, Alternaria tenuis, Cladosporium herbarum, Rhizopus arrhizus, and Penicillium spp. Various yeasts and bacteria have also been associated with the disease, especially Acetobacter bacteria, which give the grapes their pungent acetic acid smell (2). In June 2006, the two fungi most commonly isolated from affected berries (postveraison) were A. niger and A. carbonarius. Identification of the two species was confirmed by colony and spore morphology (1) and PCR analysis of the internal transcribed spacer (ITS) region of rDNA. Although A. niger was more common, A. carbonarius has not previously been reported as a pathogen of grape in California. Conidia of A. carbonarius were globose, dark with very rough walls, and could be distinguished from A. niger by their wall structure and larger size ([5.1] 6.0 to 7.6 [8.8] µm in diameter). Sequence analysis of the ITS region of isolates morphologically identified as A. carbonarius showed 100% similarity to known isolates of this species. To confirm pathogenicity, postveraison Red Globe berries on standing grapevines were wounded with a needle and inoculated by dipping entire clusters into A. carbonarius spore suspensions (106 conidia/ml) for 30 s. Sterile water was used as a control. Twenty berries on each cluster were wounded and 10 clusters were used for each treatment. The experiment was repeated in two different vineyards in Kern County in 2006 and 2007. After 48 h, water-soaked lesions could be seen on the wounding site of grapes inoculated with A. carbonarius. After 1 week, 100% of the inoculated grapes exhibited dark, black sporulation, and after 3 weeks, the clusters were almost completely rotted. Koch's postulates were completed by isolating A. carbonarius from the affected berries. A few (less than 5%) of the wounded control berries also exhibited black sporulation and rot after 3 weeks. These infections were probably the result of natural inoculum at the sites because spore traps placed in the vineyards also contained A. carbonarius and A. niger propagules. Furthermore, soil surveys in the same vineyards showed that both A. niger and A. carbonarius were present on plant debris on the vineyard floor and in the soil. To our knowledge, this is the first report of A. carbonarius causing sour rot of grapes in California. References: (1) M. A. Klich. Identification of Common Aspergillus Species. Centraalbureau voor Schimmelcultures, Urecht, The Netherlands, 2002. (2) J. J. Marois et al. Bunch rots: Miscellaneous secondary invaders and sour rot. Page 69 in: Grape Pest Management. 2nd ed. The Regents of the University of California, Division of Agriculture and Natural Resources, Oakland, 1992.

Susceptibility of Grapevine Pruning Wounds and Symptom Development in Response to Infection by Phaeoacremonium aleophilum and Phaeomoniella chlamydospora.

Fresh to 10-month-old pruning wounds on grapevine cvs. Thompson Seedless and Cabernet Sauvignon were inoculated with Phaeoacremonium aleophilum or Phaeomoniella chlamydospora at approximately 1 × 106 spores/ml. Successful infection was determined by isolation of the pathogen from necrotic margins of cankers or from vascular discoloration assessed 4 months after each inoculation date. Disease incidence decreased as the length of time between pruning and inoculation increased; however, wounds remained susceptible for up to 4 months. Vascular discoloration was significantly less extensive in the noninoculated control than in inoculated spurs. Reduction of shoot length in both cultivars varied depending on pruning wound age at the time of inoculation. In a separate study, inoculation of fresh pruning wounds of Cabernet Sauvignon resulted in successful infection regardless of the time of pruning from February to December.

Occurrence of Togninia minima Perithecia in Esca-Affected Vineyards in California.

Togninia minima is an important pathogen causing esca and grapevine declines worldwide. Although perithecia of T. minima have been produced in the laboratory, their presence in diseased vineyards has not been shown. In our study, perithecia of T. minima were found on grapevines in the field in five California counties. Perithecia were clustered on dead vascular tissue in deep cracks along trunks and cordons or on the surfaces of decayed pruning wounds. Field-collected perithecia were characteristic of T. minima perithecia previously produced in vitro and molecular sequence analysis of the internal transcribed spacer region of the nuclear ribosomal DNA additionally confirmed their identity. Ascospores from perithecia germinated on agar medium and formed colonies typical of T. minima. This is the first report of T. minima perithecia in diseased vineyards and suggests ascospores as an additional source of inoculum for new grapevine infections.

Teleomorph Formation of Phaeoacremonium aleophilum, Cause of Esca and Grapevine Decline in California.

Phaeoacremonium is a recently described genus of the hyphomycetes and includes species associated with grapevine (Vitis vinifera) declines worldwide. Spores of Phaeoacremonium spp. have been trapped in infested vineyards, but neither asexual nor sexual fruiting structures have been observed in the field. Mating studies were carried out to determine if California P. aleophilum isolates are capable of forming a teleomorph in vitro. Sterilized grapevine shavings were placed on the surface of water agar plates with pairs of different California isolates of P. aleophilum, an isolate from the holotype of P. aleophilum, plus other related Phaeoacremonium spp. After approximately 28 to 35 days, perithecia were seen forming on wood chips and agar of many pairings. Upon maturation, fertile perithecia had gelatinous droplets of ascospores oozing from their ostioles. Successful crosses, resulting in mature perithecia, corresponded to a heterothallic mating type system. When F1 progeny were backcrossed with their parents, heterothallism was confirmed. Molecular analyses of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA from ascospore progeny and perithecia confirmed that these perithecia were the teleomorph of P. aleophilum, Togninia minima. Furthermore, 4 months after moist incubating grapevine pieces from naturally infected vineyards, mature perithecia of T. minima could be seen forming on the xylem and pith tissues, suggesting both mating types occur on the same vine.