Taxonomic revision and multi-locus phylogeny of the North American clade of Ceratocystis.

The North American clade (NAC) of Ceratocystis includes pathogenic species that infect a wide range of woody hosts. Previous phylogenetic analyses have suggested that this clade includes cryptic species and a paraphyletic C. variospora. In this study, we used morphological data and phylogenetic analyses to characterize NAC taxa, including Ceratocystis isolates causing a serious disease of almond trees in California. Phylogenetic analyses based on six gene regions supported two new species of Ceratocystis. Ceratocystis destructans is introduced as the species causing severe damage to almond trees in California, and it has also been isolated from wounds on Populus and Quercus in Iowa. It is morphologically similar to C. tiliae, a pathogen on Tilia and the most recently characterized species in the NAC. Ceratocystis betulina collected from Betula platyphylla in Japan is also newly described and is the sister taxon to C. variospora. Our six-locus phylogenetic analyses and morphological characterization resolved several cryptic species in the NAC.

Taxonomy and multi-locus phylogeny of cylindrocarpon-like species associated with diseased roots of grapevine and other fruit and nut crops in California.

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

Cryptosphaeria Dieback of Fremont Cottonwood Caused by Cryptosphaeria pullmanensis and C. multicontinentalis in California.

Fremont cottonwood is a large and tall tree native to riparian areas in the southwestern United States. It grows along streams, rivers, and wetlands and serves many ecological and socioeconomic functions. In recent years, we observed a severe decline of Fremont cottonwood trees in California. Trees showed branches dying back, with dark-brown internal discoloration and decay of the wood of twigs, branches, or trunks. Eventually, the cambium and the bark were killed, causing a canker. The fungus Cryptosphaeria pullmanensis was isolated consistently from the necrotic wood of branches and twigs. On rare occasion, C. multicontinentalis was also isolated from symptomatic wood. Therefore, we investigated the pathogenicity in Fremont cottonwood of C. pullmanensis and C. multicontinentalis. Koch's postulates were completed in saplings and both species appeared highly virulent, producing internal necrosis and staining of the wood. This study is the first to report Cryptosphaeria dieback of Populus fremontii caused by C. pullmanensis and C. multicontinentalis. Symptoms and signs of this new disease are described and illustrated.

Calosphaeria Canker of Sweet Cherry Caused by Calosphaeria pulchella in California and South Australia.

California is the second largest sweet cherry producer in the United States with annual revenues up to $200 million. The South Australian cherry industry generates about 10% of Australia's overall production with approximately 1,500 metric tons of cherries produced yearly. In California, perennial canker diseases and subsequent branch dieback are responsible for extensive damage throughout sweet cherry orchards, reducing annual yields and tree longevity. Surveys of cherry orchards and isolation work were conducted in California to identify the main canker-causing agents. Calosphaeria pulchella was the main fungus isolated from cankers, followed by Eutypa lata and Leucostoma persoonii, respectively. Preliminary surveys in cherry orchards in South Australia documented C. pulchella and L. persoonii in cankers. The pathogenicity of C. pulchella in sweet cherry was confirmed following field inoculations of 2- to 3-year-old branches. C. pulchella was able to infect healthy wood and produce cankers with as much virulence as E. lata or L. persoonii. Spore trapping studies were conducted in two sweet cherry orchards in California to investigate the seasonal abundance of C. pulchella spores. Experiments showed that rain and sprinkler irrigation were important factors for aerial dissemination. Finally, this study illustrates the symptoms and signs of the new disease Calosphaeria canker.

Host range, biological variation, and phylogenetic diversity of Eutypa lata in California.

The objectives of this study were to investigate the host range of Eutypa lata in the major grape-growing regions in California and to analyze the phenotypic variation and phylogenetic diversity of E. lata isolates. Perithecia of E. lata were found on grapevines, in apricot, almond, cherry, apple, and pear tree orchards, and on ornamentals (oleander) and native plant species (California buckeye, big leaf maple, and willow). Multigene phylogenies of ribosomal DNA internal transcribed spacer, ß-tubulin, and RPB2 genes confirmed the identity of E. lata recovered from the various host plants but also revealed sequence differences among isolates. The intraspecific phylogenetic diversity of E. lata did not correspond to geography or source of isolation, and intraspecific groups were not consistent across the different DNA phylogenies. Significant phenotypic variation also was detected among E. lata isolates, including ascospore and conidium length as well as level of aggressiveness on grapevines. Pathogenicity studies proved that all isolates were infectious to grapevine, suggesting that the native vegetation surrounding vineyards can serve as inoculum sources that may constitute an important element in the epidemiology of Eutypa dieback in grapevines.

Pathogenicity of Diatrypaceae Species in Grapevines in California.

Diatrypaceous fungi have been isolated from cankered wood of grapevines (Vitis vinifera) in California. However, the pathogenicity of these fungi and their potential to cause diseases in grapevine was unknown. A series of pathogenicity tests were conducted to determine if these fungi were grapevine pathogens. In all experiments, species of the family Diatrypaceae were re-isolated from the margins of developing lesions, although frequency of re-isolation and average length of vascular discolorations varied among isolates. Cryptovalsa ampelina, Diatrype stigma, and Eutypa leptoplaca were capable of colonizing dormant canes and causing vascular necrosis. Cryptosphaeria pullmanensis, C. ampelina, D. stigma, Diatrype whitmanensis, and E. leptoplaca infected and caused lesions in green shoots of the new vegetative growth. Diatrype oregonensis and Diatrypella verrucaeformis isolates did not produce lesions that were significantly different from those produced in the control shoots and canes. This suggests that D. oregonensis and D. verrucaeformis fungi may be saprophytic rather than pathogenic to grapevine.

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.

First Report of Calosphaeria pulchella Associated with Branch Dieback of Sweet Cherry Trees in California.

California is the second largest sweet cherry producer in the United States with approximately 10,800 ha and an average annual crop value of approximately $150 million. Perennial canker diseases constitute major threats to the cherry industry productivity by reducing tree health, longevity, and yields. During the course of summer 2006, we observed severe limb and branch dieback of sweet cherry (Prunus avium L.) in San Joaquin, San Benito, Contra Costa, and Stanislaus counties of California. Isolation from diseased branches repeatedly yielded the fungus Calosphaeria pulchella (Pers.: Fr.) J. Schröt. (1,2). Cankers and vascular necroses had developed in tree limbs and branches, generally initiating from the heart wood and later spreading into the sapwood. External symptoms of disease may be unapparent throughout the early stages of infection, particularly in large diameter shoots. Older infections often appeared as wilted leaves. Branches and trunks affected with cankers from which C. pulchella was isolated also generally bore perithecia of C. pulchella beneath the periderm. Perithecia were nonstromatic and arranged in dense, circinate groups, with elongated necks converging radially and fissuring the periderm. Asci were unitunicate, clavate, and 45 to 55 × 5 to 5.5 µm. Ascospores were allantoid to suballantoid, hyaline, and 5 to 6 × 1 µm. Colonies on potato dextrose agar (PDA) were dark pink to red in their center with a white margin. Conidia were hyaline, allantoid to oblong-ellipsoidal, and (3-) 4 to 6 (-9) × 1.5 to 2 (-2.5) µm. Identification of C. pulchella isolates also was confirmed by sequence comparison in GenBank database using the internal transcribed spacer region (ITS1-5.8S-ITS2) of the rDNA. Sequences of California isolates shared 100% similarity with C. pulchella reference isolate CBS 115999 (EU367451) (2). ITS sequences of the California isolates used in this study were deposited into GenBank (Nos. HM237297 to HM237300). Pathogenicity of four isolates recovered from the margin of active cankers was determined by branch inoculations. In December 2006, 2- to 4-year-old twigs of P. avium cv. Bing were inoculated with a 5-mm cork borer to remove bark and by placing an agar plug from the growing margin of 8-day-old colonies directly into the fresh wound, mycelium side down. Ten branches per isolate were inoculated. Ten control shoots were inoculated with noncolonized agar plugs. Inoculations were covered with vaseline and wrapped with Parafilm to retain moisture. Branches were harvested in July 2007 and taken to the laboratory to be examined for canker development, and the extent of vascular discoloration in each branch was assessed. Isolations from the edge of discolored tissue were conducted to fulfill Koch's postulates. After 8 months, C. pulchella was reisolated from 100% of the inoculated branches. Length of vascular discoloration averaged 62.5 mm in branches inoculated with the four C. pulchella isolates and 16.5 mm in the control twigs. No fungi were reisolated from the slightly discolored tissue of the controls. To our knowledge, this study constitutes the first report of C. pulchella as a pathogen of sweet cherry trees in California. References: (1) M. E. Barr. Mycologia 77:549, 1985. (2) U. Damm et al. Persoonia 20:39, 2008.

Identification of Eutypa lata by PCR-RFLP.

Eutypa lata is a vascular canker pathogen of woody plants commonly diagnosed by isolating the pathogen from infected tissue. Related fungi from the same family, the Diatrypaceae, also have been found in association with grapevine in Californian vineyards. An in situ polymerase chain reaction (PCR) method has been developed for detection of E. lata in infected wood tissue. However, our results indicate that this method also would amplify other Diatrypaceous fungi, which could potentially lead to an incorrect diagnosis. Therefore, we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay. The internal transcribed spacer (ITS)1/5.8S/ITS2 ribosomal DNA region was amplified by PCR using universal primers, and RFLP patterns were determined after digestion with AluI. The restriction profiles obtained served to distinguish E. lata from wood trunk pathogens of grapevine (Phomopsis viticola, Botryodiplodia sp., Phaeoacremonium aleophilum, and Phaeomoniella chlamydospora), Diatrypaceous fungi (Diatrype sp., Diatrypella sp., Eutypella vitis, and Eutypa leptoplaca), and Cryptovalsa sp. found on dead wood of grapevine, and other Eutypa spp. (E.petrakii var. hederae, E. astroidea, E. crustata, and E. lejoplaca), with the exception of E. armeniacae, which we regard as a synonym for E. lata, and E. laevata, which has not been found on grapevine.