S. Rooney-Latham

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.

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.

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.

Cadophora species associated with wood-decay of grapevine in North America

Cadophora species are reported from grapevine (Vitis vinifera L.) in California, South Africa, Spain, Uruguay, and Canada. Frequent isolation from vines co-infected with the Esca pathogens (Togninia minima and Phaeomoniella chlamydospora), and confirmation of its ability to cause wood lesions/discoloration in pathogenicity tests, suggest that C. luteo-olivacea is part of the trunk pathogen complex. In North America, little is known regarding the diversity, geographic distribution, and roles of Cadophora species as trunk pathogens. Accordingly, we characterized 37 Cadophora isolates from ten US states and two Canadian provinces, based on molecular and morphological comparisons, and pathogenicity. Phylogenetic analysis of three loci (ITS, translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (BT)) distinguished two known species (C. luteo-olivacea and Cadophora melinii) and three newly-described species (Cadophora orientoamericana, Cadophora novi-eboraci, and Cadophora spadicis). C. orientoamericana, C. novi-eboraci, and C. spadicis were restricted to the northeastern US, whereas C. luteo-olivacea was only recovered from California. C. melinii was present in California and Ontario, Canada. Morphological characterization was less informative, due to significant overlap in dimensions of conidia, hyphae, conidiophores, and conidiogenous cells. Pathogenicity tests confirmed the presence of wood lesions after 24 m, suggesting that Cadophora species may have a role as grapevine trunk pathogens.

Occurrence of Togninia fraxinopennsylvanica on Esca-Diseased Grapevines (Vitis vinifera) and Declining Ash Trees (Fraxinus latifolia) in California

Esca (black measles) and Petri disease (young esca) are two of the most destructive diseases of grapevines in California and other grape-producing countries. This disease is now known to be caused by multiple species of Phaeoacremonium including P. aleophilum, P. angustius, P. parasiticum, P. rubrigenum, and P. mortoniae. The teleomorph of P. aleophilum was confirmed recently as Togninia minima (Tul. & C. Tul.) Berl. (2), but the teleomorph for the other Phaeoacremonium species are not known. During the summer of 2004, plant specimens were collected from declining ash trees surrounding vineyards as well as from grapevines that were showing typical symptoms of esca including the presence of purple-to-brown spots on berries and necrosis between the veins and on margins of leaves. The specimens were cut into small pieces (15 cm), placed in plastic bags, and thoroughly sprayed with 50 ml of sterile distilled water. The specimens were soaked in water for 10 min at room temperature (25 ± 2°C). Plant materials were then removed from the water and air dried under a laminar flow hood for future examination. Each wash solution was passed through 5.0- and 0.45-μm filters attached to a sterile 35-ml syringe. The 5-μm filters removed large fragments of plant tissues and larger spores, while the 0.45-μm filters trapped spores of Phaeoacremonium spp. The 0.45-μm filters were inverted onto 1.5-ml Eppendorf tubes, and 1 ml of sterile distilled water was passed through to wash out the trapped spores. Aliquots of 200 μl were spread onto potato dextrose agar amended with 0.01% tetracycline hydrochloride (PDA-tet). Colonies of P. mortoniae were identified on the plates after 10 days. Previously wetted wood pieces (grapevine and ash) were examined for fruiting bodies with a stereomicroscope. In this study, perithecia of T. fraxinopennsylvanica were discovered for the first time on diseased grapevines in California. Perithecia were embedded in decayed vascular tissue of ash branches as well as pruning wounds, cordons, and trunks of grapevine cv. Riesling from Mendocino County and cv. Thompson Seedless from Madera County. Perithecia were black, globose to subglobose, and ranged in diameter from 183 to 276 μm. They were embedded and superficial and the lengths of the perithecial necks ranged in size from 200 to 400 μm. The asci were hyaline, clavate, and ranged in size from 16.7 to 23.2 × 4.7 to 5.6 μm. Ascospores were hyaline, ellipsoid to allantoid, and ranged in size from 3.9 to 5.5 × 1.4 to 2.0 μm. When plated onto PDA-tet media, ascospores formed colonies typical of P. mortoniae. Perithecia, asci, and ascospore dimensions as well as phylogenetic analysis of sequences from the internal transcribed spacer region confirmed that the perithecia are in fact T. fraxinopennsylvanica (Hinds) Hausner et al. (1) and that this fungus is the teleomorph of P. mortoniae. Perithecia of T. fraxinopennsylvanica were also produced in vitro by crossing compatible isolates of P. mortoniae. Furthermore, spore trapping studies show that propagules of this fungus are present as airborne inoculum in infected vineyards during rainfall events throughout the growing season. References: (1) G. Hausner et al. Can. J. Bot. 70:724, 1992. (2) L. Mostert et al. Mycologia 95:646, 2003.

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. Kochs 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.

Leaf spot of Arugula, caused by Alternaria japonica, in California.

Arugula (Eruca vesicaria subsp. sativa (Mill.) Thell. is a Cruciferous plant used for culinary purposes. From 2012 to 2013, a foliar disease seriously impacted the growth and quality of about 0.1 ha of hydroponically grown arugula at a Santa Barbara County nursery. Samples of affected arugula seedlings exhibited adaxial and abaxial symptoms of mottling with circular to oval, water soaked, dark green leaf spots, each 1 to 3 mm in diameter, and some of which coalesced. Conidia of an Alternaria sp. were observed on the foliage. Symptomatic leaf pieces were disinfested with 0.6% NaOCl, blotted dry, and plated on acidified potato dextrose agar (APDA). Cultures were incubated under near-UV lights for 24 h/day. Olivaceous-grey colonies of the same Alternaria species observed on the leaves grew after 7 days. After 21 days on carrot-piece agar (3), the fungus produced beakless conidia with longitudinal and constricted transverse septa that measured 30.0 to 69.0 × 12.5 to 20.0 μm and were borne singly or in short chains of 2 to 3 conidia. In addition, knots of dark, thick-walled micro-chlamydospores were produced by the hyphae. The fungus was identified morphologically as Alternaria japonica Yoshii (2), and the species confirmed by sequence analysis. A portion of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified using ITS1 and ITS4 primers (4). The sequence (GenBank Accession No. KJ126846) was 100% identical to the ITS rDNA sequence of an isolate of A. japonica (KC584201) using a BLASTn query. A. japonica was also detected in seeds of the lot used to grow the affected arugula crop. Pathogenicity of a single isolate was tested by inoculating four 37-day-old plants each of arugula, cabbage (Brassica oleracea L. var. capitata), and broccoli (B. oleracea L. var. botrytis L.). Inoculum was obtained from 11-day-old cultures of the isolate grown at 24°C on half-strength APDA. Half of a 2.5 cm diameter agar plug containing hyphae and conidia was ground in 2 ml of sterilized water, and the volume of water increased to 45 ml. Leaves of four plants/host species were sprayed with 3.5 to 4.0 ml of inoculum. The inoculated plants and four control plants of each species treated similarly with sterilized water were immediately incubated in a dark dew chamber at 23°C. After 72 h in the dew chamber, inoculated plants of all three hosts produced similar symptoms of wilting, water soaking, and dark green leaf spotting as the original symptomatic field plants. Conidia formed in the leaf spots on both sides of inoculated leaves. A. japonica was re-isolated from all of the inoculated plants but from none of the symptomless control plants using the method previously described. Pathogenicity tests were repeated, with similar results. Although reported in Italy in 2013 (1), to our knowledge, this is the first report of A. japonica on arugula in the United States. References: (1) G. Gilardi et al. Acta Hort. 1005:569, 2013. (2) E. G. Simmons. Page 368 in: Alternaria, An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, 2007. (3) S. Werres et al. Z. Planzenkr. Pflanzensh. 108:113, 2001. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Perithecia of Phaeoacremonium viticola on Grapevine (Vitis vinifera) and Ash Tree (Fraxinus latifolia) in California

Esca and Petri diseases on grapevine are caused by Phaeomoniella chlamydospora and species of Phaeoacremonium including P. aleophilum, P. viticola, P. angustius, P. parasiticum, P. inflatipes, P. rubrigenum, and P. mortoniae. The teleomorphs of P. aleophilum and P. mortoniae have been recently confirmed as Togninia minima(Tul. & C. Tul.) Berl. (2,3), and T. fraxinopennsylvanica (Hinds) Hausner, Eyjólfsdóttir & J. Reid, respectively (1,2,3). Teleomorphs of other Phaeoacremonium spp. have not been identified, although molecular data suggests that Phaeaoacremonium spp. are linked to the genus Togninia. Naturally infected vineyards with symptoms of esca disease were surveyed during the summer and fall of 2004. Samples were collected from declining ash trees surrounding the vineyards, as well as from symptomatic grapevine trunks, cordons, and spurs. Samples were processed as previously described (1). Sterile isolations were also made from symptomatic vascular tissue of the samples and cultured on potato dextrose agar (PDA)-tet medium. Perithecia were found on the surfaces of old pruning wounds and in the cracks of cordons and trunks on Vitis vinifera cvs. Thompson Seedless, Riesling, and Cabernet Sauvignon. Perithecia were observed on grapevines in vineyards in six of nine counties, viz. Yolo, Mendocino, El Dorado, Tulare, Madera, and Sonoma. Perithecia were also observed in dead vascular tissue of declining ash tree branches (Fraxinus latifolia) located in the vicinity of vineyards in Sonoma and Yolo counties. Perithecia were black, globose to subglobose, ranging from 160 to 215 μm in diameter, and were both embedded in and superficial on the wood tissue. Neck lengths ranged from 55 to 340 μm. The asci were hyaline, clavate, and ranged in size from 12.5 to 19.5 × 3.2 to 4.5 μm. Ascospores were hyaline, ellipsoid to allantoid, and ranged in size from 3.1 to 4.1 × 1.8 to 2.1 μm. Morphologically these perithecia resemble those of Togninia spp. When plated onto PDA-tet medium, ascospores formed colonies of P. viticola after 10 days of incubation at room temperature. Additionally, P. viticola was isolated from vascular tissue of the collected plant specimens. To our knowledge, this is the first time that P. viticola has been linked to a sexual state. References: (1) A. Eskalen et al. Plant Dis. 89:528, 2005. (2) L. Mostert et al. Mycologia 95:646, 2003. (3) S. Rooney-Latham. Plant Dis. 89:177, 2005.

Interactions between a sap beetle, sabal palm, scale insect, filamentous fungi and yeast, with discovery of potential antifungal compounds.

The multi-trophic relationship between insects, yeast, and filamentous fungi is reported on sabal palm (Sabal palmetto (Walter) Lodd. ex Schult. & Schult. f.). Gut content analyses and observations of adult and larval feeding of the sap beetle Brachypeplus glaber LeConte indicate that niche partitioning of fungal food substrata occurs between adults and larvae. This is the first report of specific mycophagous niche partitioning among beetle life stages based on gut content analyses. Fungi isolated from the beetle gut of adults, larvae, and pupae include species of Fusarium Link, Cladosporium Link, and Penicillium Link, which were differentially ingested by larvae and adults; Fusarium solani and Penicillium species in larvae, whereas F. oxysoproum, F. verticillioides, and Cladosporium in adults. These data indicate the first species-level host data for Brachypeplus Erichson species. Fusarium proliferatum (Matsush.) Nirenberg was the most commonly occurring fungal gut component, being isolated from the palm as well as gut of larvae, pupae, and adults; representing a commonly shared food resource. One species of yeast, Meyerozyma caribbica (Vaughan-Mart. et al.) Kurtzman & Suzuki (basionym = Pichia caribbica), was isolated from all life stages and is likely responsible for anti-fungal properties observed in the pupae and represents a promising source of antifungal compounds; rearing and diagnostic protocols are provided to aid biomedical researchers. Feeding and cleaning behaviors are documented using time-lapse video-micrography, and discussed in a behavioral and functional morphological context. Adults spent long periods feeding, often >1/3 of the two-hour observation period. A generic adult body posture was observed during feeding, and included substrate antennation before and after ingestion. Adult grooming behaviors were manifested in distinct antennal and tarsal cleaning mechanisms. Larval behaviors were different from adults, and larvae feeding on Fusarium fungi immediately ceased all subsequent feeding. This is the first ethogram for any adult or larval sap beetle.

Entyloma helianthi: identification and characterization of the causal agent of sunflower white leaf smut

ABSTRACT White leaf smut is a minor foliar disease of sunflower (Helianthus annuus) in the United States. The disease occurs primarily in greenhouse-grown sunflowers in California and causes leaf spot, defoliation, and a reduction in yield and crop value. Historically, many Entyloma specimens with similar morphological characters, but infecting diverse plant genera including Helianthus, were called Entyloma polysporum. Recent comparative morphological and molecular work has shown that Entyloma species infect hosts within a single genus or species, suggesting that the sunflower Entyloma species may not be E. polysporum. In 2015, sunflower leaf smut material was collected from ornamental sunflowers in a greenhouse in Santa Barbara County, California. Morphologically, this species differed from E. polysporum in having smaller, more regular-shaped teliospores and prominently developed conidiophores with cylindrical conidia. The rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS]) region of the sunflower leaf smut was phylogenetically distinct from all previously sequenced Entyloma species and found only on H. annuus. This study confirms that the sunflower leaf smut pathogen represents a novel species, Entyloma helianthi. Possible misidentification of the anamorphic stage of Entyloma helianthi as another leaf spot pathogen, Ramularia helianthi, is also discussed.