First report of Cryptostroma corticale causing sooty bark disease in California and first worldwide report of silver maple as a host.

In November 2022 and February 2023, CAL FIRE tree health experts examined four maples (Acer spp.) planted decades earlier in a residential setting in Elk Grove, Sacramento Co., California (USA). Three of the trees were silver maples (Acer saccharinum ) and one was a Norway maple (A. platanoides); both species are exotic to California. The trees were in an irreversible state of decline, with the canopy substantially thinned and browning. Extensive bark cankers elongating longitudinally along the stem were visible on all trees (Fig. S1). Cankers were filled by fungal stromata protruding through the bark and producing masses of elliptical dark brown conidia (Fig. S2), approximately 5.5 x 3.7 um in size, giving the cankers a sooty appearance. The cankered bark could be peeled off easily, revealing dark and discrete lesions in the phloem and xylem. Samples from the three trees were shipped to the U.C. Berkeley Forest Pathology and Mycology Laboratory and to the CDFA PPDC in Sacramento, CA. In the laboratories, small wood chips were taken from the margins of the lesions, surface sterilized by dipping them for 30 seconds in 70% Ethanol, rinsed for 30 seconds in sterile water and plated onto 2.5% Malt Extract Agar amended with 0.3g/L Streptomycin or onto one-half strength acidified potato dextrose agar (APDA). Two morphologically identical cultures were obtained, one (T2) from a silver maple and one (T6) from the Norway maple. Cultures were then grown in liquid 2.5% malt extract broth and, after one week, DNA was extracted using the Qiagen Plant DNeasy DNA extraction kit. The ITS sequences are diagnostic for this fungus (Li et al. 2021) and those of the two cultures (GB OR064033 and OR933565) were 100% homologous to GenBank sequences of Cryptostroma corticale ( e.g. GB OP474010-11). The RPB2 sequence of T2 ( GB OR992132) was 100% homologous to that of C. corticale (GB HG934116.1). The isolate obtained from silver maple was inoculated in four potted silver maples by removing a bark disk 50 mm in diameter with a cork borer in three spots staggered at different heights and sides on the stem, placing a colonized agar plug of C. corticale in contact with the phloem, replacing the bark disk and wrapping with parafilm. Two control trees were mock inoculated using sterile agar plugs. Trees were in 57 L pots, had an average stem caliper of 2.7 cm, an average height of 3.5 m and were kept in a lath house at average high temperatures of 18-24 degrees C. After ten weeks, average lesion length was 15.4 cm (SE= 4.6) and 4.3 cm (SE=2.3) in the fungus-inoculated and control trees, respectively. An ANOVA test, nesting lesions sizes within tree, determined lesions lengths were different between inoculated and mock trees (P= 0.04). The fungus was reisolated from all points in all inoculated trees but never from control trees. C. corticale was first described in the UK from sycamore maple (Acer pseudoplatanus) (Gregory et al. 1949) and is an emerging problem in Europe (Muller et al. 2023). In North America, it has been reported from A. negundo, A. campestre, A. macrophyllum and Cornus nuttallii (Worral 2020), and it appears to be present in the Pacific Northwest (Brooks et al. 2023, Goree 1969). Norway maple is included in the European Plant Protection Organization list of hosts for C. corticale (EPPO 2023), however our finding of C. corticale on silver maple is a first report of this host worldwide and of this pathogen in California. This report is noteworthy, given that C. corticale is also a human pathogen infecting the respiratory system (Braun et al. 2021).

First Report of Phytophthora ramorum Causing Leaf Spots on Cornus capitata (Evergreen Dogwood) in United States.

Cornus capitata Wall. ex Roxb. (evergreen dogwood) is a bushy evergreen tree or shrub native to East Asia grown for its showy creamy bracts in late spring followed by attractive red fruit. In Feb 2023, a sample of foliage with leaf spots and tip dieback from C. capitata 'Mountain Moon' was submitted from a Humboldt Co. nursery as part of a CDFA inspection program for Phytophthora ramorum. The leaf spots were medium to dark brown, irregularly shaped, and ranged from 5 to 8 mm in diameter. They were located primarily along the leaf midrib and covered up to 1/4 of the leaf surface. Six 6-mm-diameter leaf discs taken from the margins of brown lesions and tip dieback were plated on Phytophthora selective CMA-PARP (PARP) media (Jeffers and Martin 1986). After 6 to 10 days, colonies resembling P. ramorum, with coralloid coenocytic hyphae, chlamydospores, ellipsoidal semi-papillate and caducous sporangia, and a relatively slow growth rate were recovered. Abundant sporangia formed on agar singly or in clusters on sympodially branched sporangiophores (n = 50), varying in size from 35 to 60 µm × 20 to 30 µm (mean 45.6 × 24.8 µm) with a length/breadth ratio ranging from 1.3 to 2.3 (mean 1.8). Chlamydospores (n = 50) ranged from 35 to 62 µm in diameter (mean 51.9 µm) on 14-day-old PARP cultures. The internal transcribed spacer region (ITS) using primers ITS5/ITS4 (White et al. 1990; accession no. OR636225) and cytochrome oxidase subunit 1 region (cox1) using primers OomCox1Levup/Fm85mod (Robideau et al. 2011; OR635665) of one isolate (0254-32A) were amplified and sequenced. BLAST analysis showed 100% identity of both regions to P. ramorum ex-type strain (MG865581 and MH136973). Microsatellite loci placed the P. ramorum isolate in the NA2 clonal lineage (Goss et al, 2011). Pathogenicity of P. ramorum isolate 0254-32A was tested using five C. capitata plants (2.5-year-old, 28-cm-tall, 3.78-liter pot). Zoospore inoculum was produced as described in Blomquist et al. (2021). Above ground parts of each plant were sprayed with inoculum (15 ml, 1.3 × 105 zoospores/ml). Inoculated plants were incubated in a dew chamber in the dark at 23°C for 72 h and then placed in a 23±1°C growth chamber with a 12-h photoperiod. Five control plants were treated as above but with sterile water instead of the zoospore suspension. Two days after inoculation, brown spots were visible on leaves on all inoculated plants, initiating from where the drops of inoculum had persisted. After 3 days, brown lesions, from water drop- to majority of entire leaf-sized, were observed on approximately 75% of inoculated leaves. After 6 days, lesions expanded to the edges of leaves, causing leaf curling and defoliation. Lesions stopped expanding after 3 weeks, and by 4 weeks, most infected leaves had abscised, with no new infections observed. Phytophthora ramorum was consistently isolated from foliar lesions of inoculated plants on PARP. It was not isolated from leaf or stem tissues of control plants, which remained asymptomatic during the 4-week experiment period. Phytophthora ramorum was detected on C. capitata in the UK in 2015 (DEFRA 2015). To our knowledge, this is the first report of P. ramorum infecting C. capitata in the United States and the completion of Koch's postulates on any Cornus spp. Incidence on C. capitata in the California nursery was low. However, their proximity to other infected foliar hosts suggests Cornus spp. may present a potential risk for the spread of P. ramorum.

First report of Phyllactinia chubutiana causing powdery mildew on Goji berry plants (Lycium barbarum and L. chinense) in the United States.

Goji berries, both Lycium barbarum, and L. chinense, are native to Asia and have been highly valued for food and medicinal purposes for more than 2,000 years (Wetters et al. 2018). These species are difficult to distinguish due to the extensive cultivar development of the former and the plasticity of the latter's phenotypes. During the summers (from July to September) of 2021 and 2022, powdery mildew was observed in Goji berry plants (L. barbarum and L. chinense) in both community and residential gardens, in Yolo Co., California. Disease severity varied between 30 and 100% of infected leaves per plant. Host identity was confirmed by phylogenetic analysis using sequences of the psbA-trnH intergenic region (Wetters et al. 2018). Powdery mildew was characterized by the presence of white fungal colonies on both sides of the leaves and the fruit sepals. Colorless adhesive tape mounts of the fungal structures were examined in drops of 3% KOH. Epidermal strips of infected leaves were peeled off for analysis of the mycelia. Hyphae were both external and internal, hyaline, septate, branched, smooth, and 2.5 to 5.8 (4.3) µm wide (n = 50). Appressoria were nipple-shaped to irregularly branched and solitary or opposite in pairs. Conidiophores were hyaline, erect, and simple. Foot cells were cylindrical, straight, 13.1 to 48.9 (29.8) × 5.0 to 8.2 (6.8) µm (n = 20), followed by 0 to 2 cells. Conidia lacked fibrosin bodies, were borne singly, unicellular, hyaline, and ellipsoid when young. Mature conidia were either cylindrical or slightly centrally constricted to dumb-bell-like, and 36.2 to 51.8 (44.9) × 15.1 to 22.0 (18.9) µm (n = 50), with conspicuous subterminal protuberances. Germ tubes were subterminal, either short with multilobate apex or moderately long with a simple end. Chasmothecia were not observed. Morphologically the fungus matched the description of Phyllactinia chubutiana Havryl., S. Takam. & U. Braun (Braun and Cook, 2012). The pathogen identity was further confirmed by amplifying and sequencing the rDNA internal transcribed spacer (ITS) and the 28S rDNA gene using the primer pairs ITS1/ITS4 (White et al. 1990) and PM3/TW14 (Takamatsu and Kano 2001, Mori et al. 2000). The resulting sequences (GenBank OP434568 to OP434569; and OP410969 to OP410970) were compared with the NCBI database using BLAST, showing 99% similarity to the ex-type isolate of P. chubutiana (BCRU 4634, GenBank AB243690). Maximum parsimony phylogenetic analysis clustered our isolates with reference sequences of P. chubutiana from various hosts deposited in GenBank. Pathogenicity was confirmed by inoculating two two-year-old L. barbarum potted plants. Four leaves per plant were surface disinfected (75% ethanol, 30 s) before gently rubbing powdery mildew infected leaves onto healthy leaves. Healthy leaves were used for mock inoculations. All plants were maintained in a growth chamber at 22°C and 80% relative humidity (RH) for five days and then 60% RH thereafter. Inoculated leaves developed powdery mildew symptoms after 28 days, and P. chubutiana colonies were confirmed by morphology, hence fulfilling Koch's postulates. Control leaves remained symptomless. Phyllactinia chubutiana (= Oidium insolitum, Ovulariopsis insolita) was first described on L. chilense in Argentina (Braun et al. 2000, Havrylenko et al. 2006), and later reported on L. chinense in China (Wang Yan et al. 2016). To our knowledge, this is the first report of P. chubutiana causing powdery mildew on L. barbarum and L. chinense in the United States, which provides crucial information for developing effective strategies to monitor and control this newly described disease.

First detection of leaf blight and stem canker caused by Phytophthora ramorum on Brisbane box in the United States.

Brisbane box,Lophostemon confertus (Myrtaceae) is a frost tender evergreen tree planted for its upright form, large ovate leaves and attractive white flowers which bloom in the spring. In June of 2017, the Plant Pest Diagnostics Center lab received a call from an arborist who described Brisbane box street trees dying in central Sausalito, Marin Co., California. Trees ranged from containing 10% to nearly 80% dead hanging leaves. Six trees along the same street were affected. Wilted brown leaves remained attached to branchlets covered in black cankers. Some healthy branchlets had leaves with angular spots which crossed the veins and were surrounded by yellow halos. Isolations were made onto CMA-PARP (Jeffers and Martin, 1986) from the canker and leaf spot margins. A Phytophthora species resemblingPhytophthora ramorum grew on CMA-PARP media with coralloid coenocytic hyphae, chlamydospores, and ellipsoidal semi-papillate sporangia. The internal transcribed spacer region (ITS) of rDNA was amplified and sequenced using primers PHY.OO.18F and PHY.OO.28SR (Rooney-Latham, et al. 2019). BLAST analysis of 770 base pairs of the sequenced amplicon (GenBank MK993541) showed 100% identity with the ITS sequence of the P. ramorum ex-type (MG865581). A portion of the cox2 gene was amplified and sequenced (Hudspeth et al. 2000) (GenBank MK994528) and 530 base pairs matched with 100% identity GU222130. Pathogenicity was confirmed by inoculating 3, initially 1.8-meter-tall trees in 18.9-liter pots. Prior to inoculations, trees were cut so they would fit into 122 cm high dew and growth chambers. For each tree, 3 lower branchlets measuring from 4 to10 mm in diameter were inoculated by wounding with a 6 mm punch, placing a colonized agar plugs in the wound, then wrapping with Parafilm. Lower branches were covered in plastic to protect them from subsequent zoospore inoculation. Branchlet inoculum was prepared by growing P. ramorum on V8 juice agar (V8) for 4 days at 22°C. Zoospores were prepared for leaf inoculation by taking 6 mm agar plugs from the margin of 6-day old cultures and flooding plugs in soil water for four days. Zoospores were released by transferring plugs to sterile distilled water at 4°C for 1.5 h. Leaves on the same three trees that were inoculated with the plugs were sprayed with 350 mL of zoospores (2 × 105 zoospores/mL), and placed in a dew chamber at 23°C for 48 h. Afterwards, they were transferred to a growth chamber (23°C, 12-h diurnal cycle) where the plastic was removed from the lower branches after leaves had dried. A single control tree was treated similarly with uncolonized V8 plugs, followed by a water spray. Leaf spots were visible 4 days later, with inoculated leaves turning necrotic and abscising after 3 weeks. Cankers from inoculated branchlets measured from 12 to 60 mm long after 60 days. Phytophthora ramorum was isolated from the margin of every inoculated canker and leaf spot. No P. ramorum was isolated from the control tree. To our knowledge, this is the first report of P. ramorum on L. confertus, in the world. Natural inoculum presumably came from infected Umbellularia californica trees located less than 800 m west of the trees in Sausalito. This detection will further limit the planting choices of arborists and landscapers in P. ramorum infected locations.

Multi-locus phylogeny and taxonomy of an unresolved, heterogeneous species complex within the genus Golovinomyces (Ascomycota, Erysiphales), including G. ambrosiae, G. circumfusus and G. spadiceus.

BACKGROUND: Previous phylogenetic analyses of species within the genus Golovinomyces (Ascomycota, Erysiphales), based on ITS and 28S rDNA sequence data, revealed a co-evolutionary relationship between powdery mildew species and hosts of certain tribes of the plant family Asteraceae. Golovinomyces growing on host plants belonging to the Heliantheae formed a single lineage, comprised of a morphologically differentiated complex of species, which included G. ambrosiae, G. circumfusus, and G. spadiceus. However, the lineage also encompassed sequences retrieved from Golovinomyces specimens on other Asteraceae tribes as well as other plant families, suggesting the involvement of a plurivorous species. A multilocus phylogenetic examination of this complex, using ITS, 28S, IGS (intergenic spacer), TUB2 (beta-tubulin), and CHS1 (chitin synthase I) sequence data was carried out to clarify the discrepancies between ITS and 28S rDNA sequence data and morphological differences. Furthermore, the circumscription of species and their host ranges were emended. RESULTS: The phylogenetic and morphological analyses conducted in this study revealed three distinct species named, viz., (1) G. ambrosiae emend. (including G. spadiceus), a plurivorous species that occurs on a multitude of hosts including, Ambrosia spp., multiple species of the Heliantheae and plant species of other tribes of Asteraceae including the Asian species of Eupatorium; (2) G. latisporus comb. nov. (≡ Oidium latisporum), the closely related, but morphologically distinct species confined to hosts of the Heliantheae genera Helianthus, Zinnia, and most likely Rudbeckia; and (3) G. circumfusus confined to Eupatorium cannabinum in Europe. CONCLUSIONS: The present results provide strong evidence that the combination of multi-locus phylogeny and morphological analysis is an effective way to identify species in the genus Golovinomyces.

Characterization and Pathogenicity of Botryosphaeriaceae Fungi Associated with Declining Urban Stands of Coast Redwood in California.

Coast redwood (Sequoia sempervirens) is among the most widely planted landscape trees in California (CA) but is in decline outside its natural range due to factors including prolonged drought and plant pathogens. We investigated associations of Botryosphaeriaceae fungi with declining coast redwood trees throughout CA. More than 100 samples were collected from 11 coastal and inland locations in CA. Fifty-nine Botryosphaeria-like fungal strains were isolated and 18 were selected for further study. Phylogenetic analysis of ITS and EF-1α sequence data confirmed the presence of Botryosphaeria dothidea, Neofusicoccum australe, N. luteum, N. mediterraneum, and N. parvum. Pathogenicity testing showed that although the Neofusicoccum species vary in virulence, all are more virulent that B. dothidea. N. australe caused the largest lesions, followed by N. luteum, N. parvum, and N. mediterraneum. Of the species recovered, only B. dothidea has been previously confirmed as a pathogen of coast redwood in CA. These results confirm that multiple Botryosphaeriaceae species are associated with branch decline and dieback on coast redwood in CA, which agrees with similar studies on woody agricultural crops. Accurate diagnosis of fungal pathogens of coast redwood is important for the development of disease management strategies and may help improve horticultural practices in maintenance of urban stands.

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

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.

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.

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.