ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
