First Report of Fusarium annulatum Associated with Young Vine Decline in California.

From 2018 to 2021 a decline was detected in young vineyards of both wine and table grape (Vitis vinifera L.) in seven counties across California (Kern, Monterey, Napa, Sonoma, Tulare, Yolo, and Yuba). Affected vines showed poor or no growth throughout the season, dieback, sap exudation and internal cankers around the graft union. Lack of feeder roots was detected, indicating weak root development. In some cases, graft failure was associated with the symptomatology in recently established vineyards (<3 years old). A prevalence from 5 to 50% was estimated in 10 vineyards. Affected vines (n=34) were collected by farm advisors and submitted to the laboratory. Symptomatic vines were surface disinfected with 70% ethanol for 1 minute and air dried under sterile conditions. Vascular discoloration around the graft union was observed and inspected by removing the bark using a sterile knife. Isolations were performed from the margin of lesions by placing five wood sections (1×1 mm) per vine onto potato dextrose agar acidified with 0.5 mL/L of 85% lactic acid (APDA) and incubated for 7 days at 25°C in the dark. Even though other fungi associated with young vine decline were isolated and identified as Phaeoacremonium, Ilyonectria, and Botryosphaeriaceae species, Fusarium colonies (Leslie and Summerell, 2006) were the most prevalent among all the symptomatic vines. Pure cultures were obtained by transferring single hyphal tips onto fresh PDA. After 5 days of incubation, colonies formed white aerial mycelium with orange to purple colors on the bottom. Colonies in Spezieller Nährstoffarmer agar (SNA) produced abundant microconidia that were hyaline and ovoid to elliptical, ranging from 5.4 to 10.6 (7.4) × 1.4 to 3.3 (2.4) µm in size (n=50). Straight and slightly curved macroconidia varied from 15.5 to 42.3 (23.7) × 2.6 to 5.0 (3.6) µm in size (n=50). Upon DNA extraction, the translation elongation factor 1α (tef1) and the RNA polymerase II second largest subunit (rpb2) partial gene regions were amplified and sequenced using the EF1/EF2, 5F2/7cR and 7cF/11aR pair primers, respectively (O'Donnell et al. 1998, O'Donnell et al. 2007, Liu et al. 1999). Consensus sequences were compared to the NCBI database using BLAST, showing over 99% similarity with the ex-type sequence of F. annulatum CBS 258.54 (MT010994 and MT010983). A maximum likelihood multi-locus phylogenetic analysis confirmed that all the Californian isolates cluster with F. annulatum strains. Sequences were deposited in GenBank (nos. OK888534 to OK888537). Two representative isolates (UCD9188 and UCD9416) were used for pathogenicity tests. One-year-old 'Chardonnay' vines were inoculated between the second and third node by removing a 5-mm diameter disk of the bark using a sterile cork borer and placing a 5-mm agar plug with actively growing mycelium. Five replicates per isolate including controls with sterile agar plugs were incubated under greenhouse conditions for 2 months. The experiment was performed twice. Symptoms expressed as vascular linear necrotic lesions that ranged from 25.6 to 62.8 mm and the same pathogen was recovered, thus fulfilling Koch's postulates. Fusarium annulatum Bugnic. is a morphologically and genetically diverse species that has been widely known as F. proliferatum and known to be pathogenic in more than 200 plant hosts (Yilmaz et al. 2021). Fusarium spp. have been previously reported to cause young vine decline in Australia and British Columbia, Canada (Highet and Nair, 1995; Úrbez-Torres et al. 2017). To the best of our knowledge, this is the first report of F. annulatum associated with young vine decline complex in California.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Ultralow oxygen treatment for control of Planococcus ficus (Hemiptera: Pseudococcidae) on grape benchgrafts.

Controlled atmosphere treatments with ultralow oxygen (ULO treatments) were developed successfully for control of vine mealybug, Planococcusflcus Signoret (Hemiptera: Pseudococcidae), on dormant grape (Vitis spp.) benchgrafts. At 30 ppm oxygen, 3-d ULO treatment at 25 degrees C and 4-d ULO treatment at 150C achieved complete control of all life stages of P. ficus. At a much lower oxygen level (<1 ppm), the two ULO treatments with the same exposure periods of 3 d at 25 degrees C and 4 d at 15 degrees C were tested on six table and wine grape cultivars grafted on rootstocks along with P. ficus. The benchgrafts were then potted in a greenhouse, together with untreated controls, to determine treatment effects on rootstock viability. Both ULO treatments achieved complete control of P. ficus and did not have any negative effects on vine growth, compared with the control. Results indicate that ULO treatments can be used to control P. ficus on dormant grape benchgrafts. The advantages of the ULO treatments are also discussed with respect to hot water treatments.

Ultralow oxygen treatment for control of Latrodectus hesperus (Araneae: Theridiidae) on harvested table grapes.

The spider Latrodectus hesperus Chamberlin & Ivie (Araneae: Theridiidae) was subjected to low and ultralow oxygen (ULO) treatments at different temperatures. Complete control of the spiders was achieved in 24-h ULO treatments with 0.5% O2 or lower at 1 degrees C and in a 24-h low oxygen (2%) treatment at 15 degrees C. Oxygen level and temperature greatly affected spider mortality. At 1 degrees C, as oxygen level was decreased from 2 to 0.5%, spider mortality increased from 0 to 100%. At 2% O2, as temperature was increased from 1 to 15 degrees C, spider mortality increased from 0 to 100%. Grape clusters from two table grape (Vitis spp.) cultivars, 'Thompson Seedless' and 'Flame Seedless', were subjected to the 24-h ULO treatment with 0.5% O2 at 1 degrees C. The ULO treatment had no negative effects on grape quality. Because of the relatively short treatment time, effectiveness at low storage temperature and the easily attained oxygen level, we conclude that the ULO treatment have good potential to be implemented commercially for control of black widow spiders on harvested table grapes.

Weed control and cover crop management affect mycorrhizal colonization of grapevine roots and arbuscular mycorrhizal fungal spore populations in a California vineyard.

Arbuscular mycorrhizal (AM) fungi naturally colonize grapevines in California vineyards. Weed control and cover cropping may affect AM fungi directly, through destruction of extraradical hyphae by soil disruption, or indirectly, through effects on populations of mycorrhizal weeds and cover crops. We examined the effects of weed control (cultivation, post-emergence herbicides, pre-emergence herbicides) and cover crops (Secale cereale cv. Merced rye, x Triticosecale cv.Trios 102) on AM fungi in a Central Coast vineyard. Seasonal changes in grapevine mycorrhizal colonization differed among weed control treatments, but did not correspond with seasonal changes in total weed frequency. Differences in grapevine colonization among weed control treatments may be due to differences in mycorrhizal status and/or AM fungal species composition among dominant weed species. Cover crops had no effect on grapevine mycorrhizal colonization, despite higher spring spore populations in cover cropped middles compared to bare middles. Cover crops were mycorrhizal and shared four AM fungal species (Glomus aggregatum, G. etunicatum, G. mosseae, G. scintillans) in common with grapevines. Lack of contact between grapevine roots and cover crop roots may have prevented grapevines from accessing higher spore populations in the middles.