The Population of Fusarium oxysporum f. sp. fragariae, Cause of Fusarium Wilt of Strawberry, in California.

The objectives of this study were to investigate the structure of the population of Fusarium oxysporum f. sp. fragariae in California and to evaluate methods for its detection. Fifty-nine isolates of F. oxysporum f. sp. fragariae were obtained from diseased strawberry plants and their identity was confirmed by pathogenicity testing. The full nuclear ribosomal intergenic spacer (IGS) and elongation factor 1-α gene (EF-1α) were amplified by polymerase chain reaction (PCR) and sequenced to elucidate phylogenetic relationships among isolates. IGS and EF-1α sequences revealed three main lineages, which corresponded to three somatic compatibility groups. Primers designed to detect F. oxysporum f. sp. fragariae in Japan amplified a 239-bp product from 55 of 59 California isolates of F. oxysporum f. sp. fragariae and from no nonpathogenic isolates of F. oxysporum. The sequence of this PCR product was identical to the sequence obtained from F. oxysporum f. sp. fragariae isolates in Japan. Intensive sampling at two locations in California showed results of tests based on PCR and somatic compatibility to be in agreement for 97% (257 of 264) of isolates tested. Our findings revealed considerable diversity in the California population of F. oxysporum f. sp. fragariae, and indications that horizontal gene transfer may have occurred.

Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew.

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Angular Leaf Spot of Cucurbits is Associated With Genetically Diverse Pseudomonas syringae Strains.

Angular leaf spot of cucurbits is generally considered to be caused by Pseudomonas syringae pv. lachrymans. It has a worldwide distribution and has been observed to emerge sporadically under humid and wet conditions. Reports of multiple P. syringae pathovars associated with the disease and lack of molecular analysis has left the true diversity of populations in the United States unclear. In this study, we collected 27 P. syringae strains causing foliar lesions and blighting on watermelon, cantaloupe, and squash in Florida, Georgia, and California over several years. Strains were fluorescent on King's medium B agar and displayed the typical phenotypic and biochemical characteristics of P. syringae. P. syringae pv. lachrymans is a member of genomospecies 2. However, the genetic profiles obtained through both MLSA (gyrB, rpoD, gapA, and gltA) and BOX-PCR (BOXA1R) identified 26 of the P. syringae strains to be distributed among three clades within genomospecies 1, and phylogenetically distinct from genomospecies 2 member P. syringae pv. lachrymans. A novel MLSA haplotype of the pathogen common to all states and cucurbit hosts was identified. Considerable genetic diversity among P. syringae strains infecting cucurbits is associated with the same disease, and reflects the larger ecological diversity of P. syringae populations from genomospecies 1.

Frequency of Verticillium Species in Commercial Spinach Fields and Transmission of V. dahliae from Spinach to Subsequent Lettuce Crops.

Verticillium wilt caused by V. dahliae is a devastating disease of lettuce in California (CA). The disease is currently restricted to a small geographic area in central coastal CA, even though cropping patterns in other coastal lettuce production regions in the state are similar. Infested spinach seed has been implicated in the introduction of V. dahliae into lettuce fields but direct evidence linking this inoculum to wilt epidemics in lettuce is lacking. In this study, 100 commercial spinach fields in four coastal CA counties were surveyed to evaluate the frequency of Verticillium species recovered from spinach seedlings and the area under spinach production in each county was assessed. Regardless of the county, V. isaacii was the most frequently isolated species from spinach followed by V. dahliae and, less frequently, V. klebahnii. The frequency of recovery of Verticillium species was unrelated to the occurrence of Verticillium wilt on lettuce in the four counties but was related to the area under spinach production in individual counties. The transmission of V. dahliae from infested spinach seeds to lettuce was investigated in microplots. Verticillium wilt developed on lettuce following two or three plantings of Verticillium-infested spinach, in independent experiments. The pathogen recovered from the infected lettuce from microplots was confirmed as V. dahliae by polymerase chain reaction assays. In a greenhouse study, transmission of a green fluorescence protein-tagged mutant strain of V. dahliae from spinach to lettuce roots was demonstrated, after two cycles of incorporation of infected spinach residue into the soil. This study presents conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce.

First Report of Stem Blight of Blueberry in California Caused by Neofusicoccum parvum.

In July 2013 in coastal (Santa Barbara County) California, commercial plantings of southern highbush blueberry (Vaccinium corymbosum) developed symptoms of a previously undiagnosed disease. Symptoms consisted of reddening and wilting of foliage, with leaves and small twigs later drying up. The bark of diseased branches was discolored and sunken; removal of this bark revealed a brown discoloration of the underlying wood. Approximately 5% of the planting was affected. When placed on acidified potato dextrose agar (A-PDA), surface disinfested pieces of symptomatic wood consistently yielded one type of fungus. On A-PDA, isolates produced extensive white aerial mycelium that turned dark gray after 4 to 5 days and formed pycnidia after 21 days. Three single-spore isolates were grown on PDA for 21 days for morphological and molecular characterization. Conidia were hyaline, smooth, and ellipsoid with round apices and truncated bases. Conidia measured 13 to 20 × 5 to 7.5 µm (n = 50; mean 16.7 × 6.1 µm), with a length/width ratio of 2.73. After 25 days, conidia became biseptate with a darker middle cell. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank KJ126847 to 49), amplified using primers ITS1 and ITS4 (5), were 99% identical to the holotype isolate of Neofusicoccum parvum Pennycook and Samuels (3) by a BLAST query (GU251125). Partial sequences of the translation elongation factor 1-alpha (EF1-α) gene (KJ126850 to 52), obtained using primers EF728Fa and EF986R (5), were 99% identical to N. parvum (GU251257). To demonstrate Koch's postulates, 14-day-old colonies of the three N. parvum isolates were grown on A-PDA. Using three blueberry cultivars (Abundance, Jewel, and Snowchaser), slits were cut beneath the epidermis of branches 1 cm diameter or less; one colonized agar plug (6 mm diameter) was placed into each cut and the epidermis was resealed with Parafilm. Ten inoculations (one inoculation per branch; two branches per plant) were made for each isolate and each cultivar; inoculated plants were maintained in a greenhouse. After 10 to 14 days, leaves on inoculated branches turned red and wilted, bark above and below the inoculation sites turned brown, and vascular tissue beneath the bark was also brown. After 21 days, diseased areas became sunken. N. parvum was recovered from all inoculated branches of all cultivars and matched the characteristics of the original isolates. Control branches, inoculated with sterile agar plugs, did not develop any symptoms and N. parvum was not isolated. This experiment was repeated with similar results. Many Botryosphaeriaceae species, including N. parvum, are associated with canker and dieback symptoms on blueberry worldwide (2). To our knowledge, this is the first documentation of stem blight caused by N. parvum on blueberry in CA. Blueberry is a rapidly expanding industry in the state, with 960 ha planted in 2005 increasing to 2,830 ha in 2012 (1). Drought stress predisposes plants to stem blight caused by Botryosphaeriacease species (4); therefore, expansion into arid areas of CA could increase the incidence and severity of N. parvum. References: (1) N. Amer. Blueberry Council. 2012 World Blueberry Acreage & Prod. Rept., 2013. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., online publication, ARS, USDA. Retrieved February 5, 2014. (3) S. R Pennycook and G. J. Samuels. Mycotaxon 24:445, 1985. (4) W. A. Sinclair and H. H. Lyon. Diseases of Trees and Shrubs, Second Edition. Comstock Publ. Assoc. 2005. (5) B. Slippers et al. Mycologia 96:83, 2004.

First Report of Southern Blight of Swiss Chard (Beta vulgaris subsp. cicla) Caused by Sclerotium rolfsii in California.

In June 2013, a commercial organic planting of Swiss chard (Beta vulgaris subsp. cicla) in Monterey County, CA, showed symptoms of a soilborne disease. Early symptoms consisted of delayed and stunted growth, with wilting of foliage during the warmer times of the day. Initially, a light brown discoloration developed on stems at the soil line. As disease progressed, a dark brown necrosis extended up the main stem and down along the upper portion of the taproot. In advanced cases, the plants collapsed and died. Extensive white cottony mycelium and numerous brown, spherical sclerotia, approximately 1 mm in diameter, developed externally on the lower stem, crown, and adhering adjacent soil. For this particular planting, approximately 10% of the 0.4 ha was lost. Sequentially planted sets of chard placed in other parts of the farm were unaffected. Isolations from necrotic plant tissues, sclerotia, and white mycelium all resulted in recovery of the same white fungus that in culture produced identical sclerotia but no other reproductive structures. Based on white mycelium, sclerotia morphology, and the presence of clamp connections at hyphal septa, the fungus was identified as Sclerotium rolfsii (1). Pathogenicity was tested by growing isolates on potato dextrose agar, drying the resulting sclerotia for 48 h, then burying 5 to 8 sclerotia adjacent to the crowns of healthy Swiss chard plants grown in pots. Three isolates were tested using 24 plants per isolate. Six control plants were inoculated with sterilized sand. All plants were incubated in a greenhouse at 22 to 25°C. After 8 days, inoculated plants began to wilt. By 14 days after inoculation, 100% of the inoculated plants showed symptoms identical to those observed in the field. One half of the plants were used for re-isolations, from which S. rolfsii was recovered from all necrotic crown and stem tissues. The other half of the plants were maintained in the greenhouse; these plants later supported the development of sclerotia. Sand-inoculated control plants did not develop any disease symptoms. The experiment was repeated and the results were the same. To our knowledge, this is the first report of southern blight of Swiss chard in California. Southern blight has not previously been found in this cooler, western part of the county adjacent to the Pacific Ocean; southern blight has been documented on other crops such as pepper, tomato, and chives (3) in the warmer eastern and southern parts of Monterey County. S. rolfsii has been reported on Swiss chard in Louisiana, South Carolina, and Cuba (2). References: (1) K. H. Domsch et al. Compendium of Soil Fungi, 2nd edition. IHW-Verlag, Eching, Germany, 2007. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab. Online publication, ARS, USDA. Retrieved July 26, 2013. (3) S. T. Koike et al. Plant Dis. 78:208, 1994.

Pseudomonas Blight Caused by Pseudomonas syringae on Raspberry in California.

In February 2013 in coastal California (Santa Cruz County), plantings of red raspberry (Rubus idaeus var. strigosus) exhibited symptoms of a previously undocumented disease. Initial symptoms were small (less than 5 mm wide), angular, water-soaked lesions on leaf and petiole tissues of recently emerged foliage. Lesions were observable from both adaxial and abaxial leaf surfaces. As disease progressed, lesions enlarged and coalesced, resulting in significant dark brown to black blighting of the foliage. The foliage of severely affected plants was stunted and wilted. The disease affected 5 ha and incidence was approximately 30%. Cream-colored bacterial colonies were isolated from surface disinfested symptomatic tissue that was macerated and streaked onto King's medium B (KMB) and sucrose peptone agar (SPA). Fungi were not recovered from any tissue that was surface disinfested and placed into acidified potato dextrose agar. Four representative strains were fluorescent on KMB and gram-negative based on lysis by KOH. Strains were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices but induced a hypersensitive response in tobacco (Nicotiana tabacum L. cv. Samsun); strains thus belonged to Lelliot's LOPAT group 1, P. syringae (3). All four strains had identical DNA fragment-banding patterns generated by repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer (4). The pattern generated was different than all P. syringae pathovars in genomospecies 1 including P. syringae pv. syringae. According to multilocus sequence analysis conducted by previously described methods, the strains are most closely related to P. syringae pv. aceris and P. syringae pv. solidagae in genomospecies 1 (1). Potted raspberry plants were used to test four strains for pathogenicity. Inoculum was prepared by growing the bacteria on SPA for 48 h and suspending the bacteria in sterile distilled water (SDW) for a final concentration of approximately 107 CFU/ml. Suspensions were sprayed until runoff onto three replicate plants per strain. Control plants were sprayed with SDW until runoff. Plants were enclosed in plastic bags for 24 h and then maintained in a greenhouse (23 to 25°C). After 7 to 8 days, water soaked lesions developed on all inoculated plants; lesions later turned dark brown and appeared similar to symptoms observed in the field. Plants treated with water developed no symptoms. Bacteria re-isolated onto KMB from symptomatic tissues were fluorescent and appeared identical to the bacteria used to inoculate the plants; two selected re-isolated strains were identical to the original strains according to rep-PCR, fluorescence, and LOPAT reactions. The experiment was repeated and disease development and recovery of fluorescent strains on KMB was identical to the first experiment. To our knowledge, this is the first report of Pseudomonas blight of raspberry, caused by P. syringae, in California. Affected plants initially were stunted in growth but later in the summer exhibited no lasting effects from the disease. Pseudomonas blight has been reported in the Pacific Northwest region of the United States, the British Columbia region of Canada, and Serbia (2). References: (1) C. T. Bull et al. Phytopathology 101:847, 2011. (2) Z. Ivanovic et al. Eur. J. Plant Pathol. 134:191, 2012. (3) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966. (4) A. S. A. Marques, et al. Genet. Mol. Biol. 31:106. 2008.

First Report of White Mold Caused by Sclerotinia minor on Mexican Sunflower in California.

Mexican sunflower (Tithonia rotundifolia) is a plant in the Asteraceae that is grown commercially as a cutflower commodity and also as a beneficial insectary plant. In June 2012 in coastal California (Santa Cruz County), several fields of organic lettuce (Lactuca sativa) were interplanted with direct-seeded rows of Mexican sunflower (cv. Torch) in order to attract beneficial insects. When approximately 2 to 3 weeks from harvest, lettuce plants began to wilt and collapse. Lettuce crowns were decayed and covered with white mycelium and small (0.5 to 3 mm diameter), irregularly shaped, black sclerotia. These plants were confirmed to have lettuce drop disease caused by Sclerotinia minor (2). In addition, Mexican sunflower plants began to wilt and eventually died. Initial symptoms on crowns and bases of the main stems in contact with soil consisted of a light tan discoloration. These discolored areas turned darker brown, became necrotic, and later were covered with white mycelium and sclerotia that were identical to those found on lettuce. Symptomatic sunflower stems were surface disinfested and small pieces from the margins of necrotic areas were placed into petri plates containing acidified potato dextrose agar. Resulting fungal colonies were white, produced profuse numbers (approx. 39 sclerotia/cm2) of small black sclerotia, and were identified as S. minor. Six-week-old Mexican sunflower plants grown in a peat moss-based rooting medium in 5-cm square pots were used to test the pathogenicity of four isolates. Isolates were grown on cubed and autoclaved potato pieces and resulting sclerotia were recovered and dried (1). For each isolate, 12 plants for each of three cultivars (cvs. Fiesta del Sol, Torch, and Yellow Torch) were inoculated by placing 3 to 5 sclerotia 1 cm below the soil level and adjacent to the plant crowns/stem bases. Sterile sand was placed next to crowns of the control plants. Plants were maintained in a greenhouse at 22 to 24°C. Symptom development was rapid and after 6 to 7 days, inoculated Tithonia plants exhibited brown necrosis at inoculated areas. After 10 days, Tithonia crowns were decayed and plants wilted. S. minor was reisolated from selected necrotic crown and stem tissues. Diseased plants that were not used for reisolations later supported the growth of the characteristic white mycelium and black sclerotia. There were no significant differences between the Tithonia cultivars, and overall disease incidence ranged from 74 to 100%. Non-inoculated plants were asymptomatic. The experiment was repeated and results were similar. In addition, the sclerotia of the four Tithonia isolates were similarly inoculated onto sets of 12 romaine lettuce plants (cv. Green Towers). After 5 to 6 days, all plants developed lettuce drop disease and the pathogen was reisolated. To my knowledge, this is the first report of Mexican sunflower as a host of S. minor. These findings indicate that Mexican sunflower and lettuce are susceptible to the same lettuce drop pathogen, and that this beneficial insectary plant could increase soilborne inoculum of S. minor. Growers should therefore be aware of the host status of beneficial insectary and other plants interplanted with crops. References: (1) P. Chitrampalam et al. Phytopathology 101:358, 2011. (2) K. V. Subbarao. Plant Dis. 82:1068, 1998.

Verticillium Wilt of Spineless Safflower Caused by Verticillium dahliae in California.

Spineless selections of Carthamus tinctorius (safflower) are grown as commercial field grown cutflower crops in coastal California. In 2010, field plantings of spineless safflower in Santa Clara County developed symptoms of a wilt disease. Affected plants were stunted and slow to develop. As plants developed flower buds, lower leaves turned yellow and wilted. As disease developed, lower leaves turned tan and desiccated; in extreme cases, the entire plant wilted and died. Examination of the taproot and stem vascular tissue revealed a tan to light brown, longitudinal, vascular discoloration. Disease distribution was patchy but in some plantings up to 50% of the plants were unharvestable due to loss of quality or plant death. Isolation from symptomatic vascular tissue consistently resulted in the recovery of a fungus with white aerial mycelium, verticillate conidiophores, single-celled, ovoid to ellipsoid, hyaline conidia, and solitary black microsclerotia that were rounded to elongated or irregular in shape. Three single conidial isolates were chosen for species and race identification after DNA extraction using the FastDNASPIN Kit (MP Biomedicals, Solon, OH). Verticillium species-specific PCR amplified a 500-bp amplicon that is specific to Verticillium dahliae from all three isolates. All three isolates also amplified the race 2-specific 270-bp band in PCR. No amplification was observed in race 1-specific PCR. Based on morphological and molecular data, the fungus was identified as V. dahliae (1,3). Pathogenicity of two isolates was tested individually by soil drench inoculations using 10 ml of conidial suspensions (7 × 106 conidia/ml) for each of 10 containerized plants grown in a peat moss mix in 7.6 cm diameter pots. Five safflower selections were inoculated and maintained in a greenhouse. After 6 weeks, as plants began to form flowers, inoculated plants showed lower leaf dieback and plant wilting. Vascular discoloration was observed when plants were dissected. V. dahliae was consistently recovered from symptomatic tissue. Control plants that only received water did not develop symptoms. The experiment was repeated and the results were consistent. To our knowledge, this is the first report of Verticillium wilt of ornamental, spineless safflower. This disease has been reported previously on agronomic safflower grown as an oilseed crop (2). This finding has significance for coastal crop rotation decisions as ornamental safflower is yet another host that could augment V. dahliae soil inoculum levels for crops such as strawberry and vegetables. References: (1) P. Inderbitzin et al. PLoS One 6: e28341, 2011. (2) J. M. Klisiewicz. Plant Dis. 65:237, 1981. (3) Maruthachalam et al. Phytopathology 100:1222, 2010.

First Report of Twig Canker on Willow Caused by Colletotrichum acutatum in California.

Following prolonged spring rains and cool summer weather in 2010, mature weeping willow trees (Salix babylonica L.) growing next to a manmade lake in Marin County, CA, showed symptoms of a previously undescribed disease. During summer, small branches developed dark brown to black, sunken cankers. Canker lengths ranged from 3 to 20 cm. Within the cankered areas, affected twigs, shoots, and leaves turned brown, collapsed, and died. The distal portions of infected branches also died, giving the trees a blighted appearance. Acervuli and pink sporulation were observed in the canker tissue. When placed on acidified potato dextrose agar (A-PDA), canker tissues consistently yielded one type of fungal organism. On A-PDA, isolates produced gray aerial mycelium, acervuli, and single-celled fusiform conidia. Two isolates were identified as Colletotrichum acutatum based on sequence analysis of the ITS region of the ribosomal DNA and the 1-kb intron of the glutamine synthase gene (1) and fungal morphology (2,3) (GenBank Accession Nos. JQ951597 and JQ951598). The willow isolates examined were identified as C. acutatum based on a 99% identity of the ITS sequence with accession FR716517 and a 98% identity of the 1-kb intron sequence with accession GQ387248 in GenBank. Interestingly, the isolates were confirmed to be homothallic producing perithecia from monoconidial cultures. To demonstrate Koch's postulates, inocula were prepared from 2-week-old colonies of each of four isolates grown on A-PDA. Using containerized weeping willow trees as test material, shallow slits were cut into the epidermis of small (1.5-cm diameter or less) branches; one colonized agar plug was placed within each cut area and the epidermis was resealed by wrapping the branch with Parafilm. Ten inoculations were made for each isolate and inoculated plants were maintained in a greenhouse. After 4 weeks, inoculated branches exhibited dark cankers and twig dieback. C. acutatum was reisolated from all symptomatic cankers and matched the characteristics of the original isolates. Control twigs, inoculated with sterile agar plugs, did not develop any blight symptoms. This experiment was repeated and the results were the same. To our knowledge, this is the first documentation of C. acutatum as a pathogen of weeping willow in California. The disease resulted in repeated defoliation of trees in the Santa Venetia area of Marin County. Badly infected trees declined as a result of repeated defoliation and twig loss. Discussions with parks personnel suggested that the disease may have been present at low levels in the area for some years, and that disease severity increased dramatically with weather that was atypically wet and cool (max. mean temps. 5.5°C cooler and 6 cm more total rainfall than the records of the previous two years) for the area during May and June 2010, when the disease was discovered. References: (1) J. C. Guerber et al. Mycologia 95:872, 2003. (2) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

Bacterial Leaf Spot of Radicchio (Cichorium intybus) is caused by Xanthomonas hortorum.

Radicchio (Cichorium intybus) is ranked 22 among crops in Monterey County, California, with a farm gate value of $19,531,000 (3). Beginning in 2002, a leaf spot disease of radicchio was observed in Monterey County. The disease began as small lesions and in some cases coalesced into larger, irregular spots. Lesions were maroon to dark brown; in some cases, the margins of brown lesions became dark maroon with aging. Each leaf spot was observable from both adaxial and abaxial leaf surfaces. Symptoms primarily occurred on the outer foliage of the heads, though on occasion the head cap leaf could develop lesions. Disease incidence in the first year resulted in up to 10% unharvested radicchio because of cap leaf infections or reduced head size if outer wrapper leaves were all removed; outbreaks in subsequent seasons were more limited. Bacteria forming yellow mucoid colonies were isolated from surface disinfested symptomatic tissue that was macerated and streaked onto sucrose peptone agar medium. Bacteria were gram negative, did not fluoresce on King's Medium B, and used esculin as a carbon source but used none of the other 48 carbon sources tested using the API 50 CH test strip. Nine isolates from symptomatic radicchio had the same DNA fragment banding pattern generated by repetitive extragenic palindromic sequence polymerase chain reactions (rep-PCR) using the BOXA1R primer. Amplicons of rpoD, dnaK, fyuA, and gyrB for multilocus sequence typing (MLST) were generated using a modification of the scheme developed by Young et al. (4) and sequenced by a commercial laboratory. Concatenated sequences of the four genes from the radicchio isolates were compared to the sequences available in the Plant Associated and Environmental Microbes Database (1). The genetic distance between the nine isolates from radicchio and pathotypes of Xanthomonas hortorum were 0.03 or less and MLST analysis indicated that radicchio isolates were members of the species X. hortorum (2). To complete Koch's postulates, freshly grown cultures were suspended in phosphate buffer and adjusted to approximately 5 × 108 CFU/ml. The inoculum was sprayed onto the undersides of leaves of 40-day-old radicchio plants (C. intybus cv. Leonardo). Plants were incubated at 100% humidity for 48 h and then moved to a greenhouse. Plants sprayed with buffer served as negative controls. For each of the two experiments conducted, there were three and six single-plant replicates per treatment. The buffer treated plants did not develop symptoms but the plants treated with isolates from radicchio developed leaf spots similar to those observed in the field with symptoms beginning to be visible after 5 days. The bacteria isolated from symptomatic tissue on inoculated plants were identical to the original strains when compared with rep-PCR, thus completing Koch's postulates. Results from the two experiments were similar. To our knowledge, this is the first report of X. hortorum causing a leaf spot disease on radicchio. The disease continues to occur sporadically on radicchio grown in coastal California. References: (1) Almeida et al. Phytopathology 100:208, 2010. (2) Bull et al. Phytopathology 101:847, 2011. (3) Lauritzen, Monterey County Crop Report, 2010; (4) Young et al. Syst. Appl. Microbiol. 31:366, 2008.

Gray Mold of Green Shiso (Perilla frutescens), Caused by Botrytis cinerea, in California.

Shiso (Perilla frutescens) is a leafy herb in the Lamiaceae family and is widely used in Japanese and other Asian cuisine for cooking, pickling, oil (from the seeds), and garnish. A number of shiso types are used, though the most common are green shiso (ao-shiso) and red shiso (aka-shiso). In the winter months of 2010 and early spring 2011, a foliar blight disease developed on greenhouse-grown green shiso produced in Ventura County, CA. Initial symptoms were angular, dull green leaf lesions on older foliage. Such lesions often were initiated along leaf edges. As the disease progressed, lesions turned gray green, expanded, and could affect most of the leaf surface. Lesion tissue became dry and papery in texture; signs of a pathogen were not present. Tests for bacterial agents were negative. However, a fungus was consistently isolated from symptomatic leaves. Isolates of this fungus were grown on potato dextrose agar (PDA) in petri plates incubated under fluorescent lights and were identified as Botrytis cinerea (1). On PDA, mycelial growth was gray brown and conidiophores measured 2 mm or longer and were branched at the terminals. Conidia were aseptate, hyaline, ellipsoidal, and measured (6.5-) 8.4 to 9.2 (-12.0) × (6.1-) 6.8 to 8.0 (-9.5) µm. Sclerotia were not present. Pathogenicity of four isolates was tested by spraying conidial suspensions (1 × 105 conidia/ml) until runoff onto sets of potted green and red shiso plants. Each set consisted of six wounded (leaf tips cut) and six unwounded plants. Plants were enclosed in plastic bags for 48 h and then maintained at 22 to 24°C in a greenhouse. After 4 days, leaf lesions developed on both wounded and unwounded leaves of green and red shiso. The resulting symptoms were similar to those observed in commercial production and B. cinerea was recovered from symptomatic tissue. Non-inoculated, wounded, and unwounded red and green shiso plants were sprayed with distilled water and did not develop symptoms. This experiment was conducted two times and results were the same. To our knowledge, this is the first report of gray mold of shiso in the United States caused by B. cinerea. The disease caused significant damage to the shiso crop because symptomatic leaves are unacceptable for market. In 2010, the greenhouse facility that contained the diseased shiso had numerous leaks in the roof; winter rains that occurred during this time therefore resulted in higher free moisture and humidity in the growing area, which likely provided optimum environmental conditions for the pathogen to infect and cause disease on shiso. Reference: (1) M. B. Ellis and J. M. Waller. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 431, 1974.

First Report of Turnip mosaic virus in Tomatillo (Physalis philadelphica) in California.

Tomatillo is an important vegetable in Mexican cuisine. It is of Mesoamerica origin and now is grown widely in the Western Hemisphere. In 2011, 2% of commercially grown tomatillo plants in San Benito County, California exhibited severe stunting with foliage showing mosaic symptoms and leaf distortion. The fruits on infected plants were mottled and unmarketable. Flexuous filamentous-shaped virus particles of 800 to 850 nm long and 11 to 12 nm wide were observed from sap of the symptomatic plants with a transmission electron microscope. Sap from the diseased tomatillo plants reacted positively in an immunostrip assay for potyvirus (Agdia Inc., Elkhart, IN), indicating a potyvirus was associated with the disease. The causal agent was mechanically transmitted from the diseased field plants to six virus-free greenhouse tomatillo plants and all inoculated plants induced identical symptoms. The causal agent was also transmitted to Chenopodium quinoa and C. murale (chlorotic local lesions) and Nicotiana clevelandii, N. tabacum, and Physalis wrightii (systemic symptoms). The disease was also transmitted to tomatillo plants by the green peach aphid (Myzus persicae) in a nonpersistent manner (1-min acquisition access period and 1-min transmission access period with no latent period). To further identify the causal agent, total nucleic acids were extracted by a cetyltrimethylammoniumbromide (CTAB) method (2) and tested by reverse transcription-PCR using potyvirus degenerate primers CIFor and CIRev (1). An amplicon of approximately 700 bp from the diseased tomatillo was cloned and sequenced. Analysis of the 631-bp partial CI sequence (GenBank Accession No. JN601884) showed that the virus had 93.6% nucleotide identity and 100% amino acid identity with cognate regions of Turnip mosaic virus (TuMV) (GenBank Accession No. D10927). Our results indicated that the disease was caused by TuMV. To our knowledge, this is the first report of TuMV in tomatillo. Since TuMV has a wide host range and is readily transmitted by green peach aphids, TuMV could be a new threat to tomatillo production in California. References: (1) C. Ha et al. Arch. Virol. 153:25, 2008. (2) R. Li et al. J. Virol. Methods 154:48, 2008.

First Report of Bacterial Streak of Fennel (Foeniculum vulgare) in California Caused by Pseudomonas syringae pv. apii.

A previously undescribed disease appeared on commercially grown fennel (Foeniculum vulgare) in Salinas (Monterey County), CA in March 2010. Initial symptoms consisted of small, dark brown-to-black lesions on leaves and stems. As disease progressed, lesions expanded in a linear fashion and eventually spread down the stems and into the bulbs. Once the disease reached the fennel bulbs, the plants were unmarketable. Eighteen, gram-negative, blue-fluorescing bacterial isolates were obtained on sucrose peptone agar from individual plants of two outbreaks from different fields in Salinas. The isolates were dome shaped on sucrose-amended media and produced necrotic lesions when injected into tobacco. Isolates were oxidase negative and did not rot potatoes or hydrolyze arginine. These reactions corresponded to Lelliot's group 1, which includes Pseudomonas syringae (2). The fennel isolates were identical to recently characterized isolates of P. syringae pv. apii from parsley grown in coastal California (1) and similar to the pathotype strain of P. syringae pv. apii according to DNA fragment banding patterns of amplicons generated from repetitive extragenic palindromic sequence (rep)-PCR using the BOXA1R primer. The isolates were distinct from the pathotype strain P. syringae pv. coriandricola. Like P. syringae pv. apii, the fennel isolates did not nucleate ice but did hydrolyze gelatine. Six isolates were tested for pathogenicity in each of two independent experiments with a total of six replications per isolate. Healthy, potted fennel plant stems of a proprietary cultivar were pierced once with insect display pins that had been dipped into bacterial colonies grown for 48 h on nutrient agar. Control stems were inoculated with sterile pins. Plants were incubated for 48 h at 100% relative humidity and then held in a greenhouse. After 4 to 6 days, inoculated fennel plants developed symptoms similar to those originally observed in the field. Bacteria isolated from surface-disinfested symptomatic tissue were fluorescent and identical to strains used to inoculate plants as evaluated by rep-PCR, thus fulfilling Koch's postulates. Control plants remained symptomless. To our knowledge, this is the first report of this pathogen causing a disease on fennel; the disease has been named bacterial streak. The disease occurred in three fields in 2010 and incidence was <1% in each case. Similar symptoms were also observed on fennel in the 2011 season in at least two fields. Previously, the host range of P. syringae pv. apii was reported to be restricted to celery. This research expands the natural host range of P. syringae pv. apii; thus, care should be taken in choosing Apiaceae crops for plantings subsequent to the occurrence of this pathogen. References: (1) C. T. Bull et al. Phytopathology 101:847, 2011. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

First Report of Embellisia allii Causing Skin Blotch and Bulb Canker on Garlic in California.

In April 2011, commercial garlic (Allium sativum) in Monterey County, CA showed symptoms of an undocumented disease. Bulb and stem sheaths were dark, decayed, and sloughing off the plants. Dissection of diseased sheaths revealed black hyphae between layers. Lower leaves wilted, turned tan, and dried up. Disease occurred in small patches scattered in two fields. In the patches, disease incidence was as much as 50%; however, overall field incidence was less than 1%. Isolations from 80% (16 of 20 plants) of collected plants resulted in the recovery of a dark olivaceous black fungus. Conidiophores were geniculate and brown and conidia were borne singly, brown, and ellipsoidal to cylindrical. Conidia had two to five but mostly three transverse septa. Longitudinal septa were infrequent and apical cells were rounded. Conidia measured (19.0-) 26.3 to 36.6 (-42.8) × (6.7-) 9.2 to 9.9 (-12.9) µm. Dark, intercalary chlamydospores were observed as colonies aged. DNA sequencing of the internal transcribed spacer (ITS) regions of four, single-spored isolates was completed with primers ITS1 and ITS4 (3). Sequences of all isolates (GenBank Nos. JN588614 to JN588617) were identical and 100% similar to Embellisia allii (AY278840). On the basis of morphological and molecular data, the fungus was identified as E. allii (Campanile) Simmons (1). Pathogenicity of four of the sequenced E. allii isolates and one additional E. allii isolate was tested using inoculum grown on acidified potato dextrose agar and garlic (cv. California Late) planted into 15-cm pots. A transverse incision was made at a point 2 cm above the garlic bulb so that a colonized agar plug could be inserted between the second and third sheath layer. The stem was then wrapped with Parafilm. Ten plants per isolate were inoculated and kept in a greenhouse (24 to 26°C). Seven to eight days after inoculation, the tissue around the incision turned tan and dark fungal growth was observed. Fourteen days after inoculation, the inoculated area was necrotic and dark fungal growth developed between stem layers. E. allii was reisolated from all inoculated plants and matched the morphological characteristics of the original isolates. Control plants, inoculated with uncolonized agar plugs, developed no symptoms. This experiment was repeated with similar results. In addition, one isolate was used to inoculate leek (A. porrum cv. Lancelot) and onion (A. cepa cv. Evergreen). Similar symptoms developed on these two species and E. allii was reisolated from all plants. To our knowledge, this is the first documentation of skin blotch and bulb canker caused by E. allii on garlic in California. Affected plants were of poor quality and could not be harvested. Our findings that garlic isolates of E. allii can infect leek and onion provide preliminary evidence that this pathogen is not restricted to garlic; this information may be useful to growers when considering crop rotations. E. allii has been reported on garlic in a number of places in Africa, Asia, Europe, the Middle East, and North and South America (2). The sequenced E. allii isolates are deposited in the fungal collection at the CDFA Plant Pest Diagnostics Lab (CDFA798-801). References: (1) J. C. David. Mycopathologia 116:59, 1991. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , August 8, 2011, (3) B. M. Pryor and D. M. Bigelow. Mycologia 95:1141, 2003.

First Report of Fusarium Stem and Crown Rot of Fennel in Arizona Caused by Fusarium avenaceum.

In 2010 in Yuma, AZ, field-grown fennel (Foeniculum vulgare, Apiaceae) exhibited previously undescribed disease symptoms. The lower stems in contact with soil developed a brown decay and leaves on these stems became chlorotic. White mycelium and orange sporodochia were observed on affected tissues near the soil line. Diseased stems later wilted, died, and resulted in reduced quality of the fennel; these plants were not harvested. Disease distribution was patchy and prevalence was approximately 5%. Symptomatic tissues were surface sterilized in a dilute (1%) bleach solution for 3 min and tissues from the margins of the decay were placed into petri plates containing acidified corn meal agar (2 ml of 25% lactic acid/liter). Isolations consistently resulted in the recovery of a presumptive Fusarium species. Isolates were transferred to carnation leaf agar and incubated at 22°C under fluorescent lights for 10 days. Morphologies of all isolates were identical, with macroconidia being long and slender, slightly curved, with elongated, bent apical cells and notched basal cells. Conidia were borne on monophialides. Microconidia were sparse and chlamydospores were not observed. For two isolates, a portion of the translation elongation factor 1-alpha gene (TEF) was amplified with primers ef1 and ef2 (3). Based on a comparison of 668 base pairs, both isolates had the same sequence, which differed by one base pair from an accession (GQ915502.1) of Fusarium avenaceum in GenBank. The same single base pair also separated the two fennel isolates from an isolate of F. avenaceum (GL 13) previously recovered from Eustoma grandiflorum (=Lisianthus russellianus) (2). Thus, both morphological and molecular criteria support identification of the recovered fungus as F. avenaceum (Fries) Saccardo. Partial TEF sequences were deposited in GenBank (Accession Nos. JN254784, JN254785, and JN254786 for the two fennel isolates and GL 13, respectively). All isolates are archived in the Department of Plant Pathology at University of California, Davis. Pathogenicity was tested by cutting shallow slits into fennel stems, inserting one colonized agar plug into each cut, and wrapping the stems with Parafilm. Five isolates from fennel were tested on 10 stems each. Control plants were inoculated with uncolonized agar plugs. Plants were maintained at 24 to 26°C in a greenhouse. After 6 to 8 days, a brown decay developed on 70 to 90% of Fusarium-inoculated stems at the points of inoculation. Foliage later became chlorotic and F. avenaceum was recovered from all symptomatic stems. Control plants were symptomless. The experiment was completed two times and results were the same. In addition, F. avenaceum isolate GL13 from E. grandiflorum (2) was inoculated onto fennel plants with the same method. However, these inoculated plants remained symptomless. To our knowledge, this is the first report of a stem and crown rot disease of fennel caused by F. avenaceum. Apparently, the only other published account of a Fusarium disease of fennel is root rot caused by F. solani (1). The inability of the Eustoma isolate of F. avenaceum to cause disease in fennel suggests that these two crown rot pathogens may have restricted host ranges. References: (1) J. H. Gupta and V. P. Srivastava. Indian J. Mycol. Plant Pathol. 8:206, 1979. (2) S. T. Koike et al. Plant Dis. 80:1429, 1996. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. U.S.A. 95:2044, 1998.

Phenological and phytochemical changes correlate with differential interactions of Verticillium dahliae with broccoli and cauliflower.

Cauliflower (Brassica oleracea var. botrytis subvar. cauliflora) is susceptible to wilt caused by Verticillium dahliae but broccoli (B. oleracea var. italica subvar. cyamosa) is not. Infection of broccoli and cauliflower by a green fluorescent protein-expressing isolate of V. dahliae was examined using epifluorescence and confocal laser-scanning microscopy to follow infection and colonization in relation to plant phenology. Plant glucosinolate, phenolic, and lignin contents were also assayed at 0, 4, 14, and 28 days postinoculation. V. dahliae consistently infected and colonized the vascular tissues of all cauliflower plants regardless of age at inoculation, with the pathogen ultimately appearing in the developing seed; however, colonization decreased with plant age. In broccoli, V. dahliae infected and colonized root and stem xylem tissues of plants inoculated at 1, 2, or 3 weeks postemergence. However, V. dahliae colonized only the root xylem and the epidermal and cortical tissues of broccoli plants inoculated at 4, 5, and 6 weeks postemergence. The frequency of reisolation of V. dahliae from the stems (4 to 22%) and roots (10 to 40%) of mature broccoli plants was lower than for cauliflower stems (25 to 64%) and roots (31 to 71%). The mean level of aliphatic glucosinolates in broccoli roots was 6.18 times higher than in the shoots and did not vary with age, whereas it was 3.65 times higher in cauliflower shoots than in the roots and there was a proportional increase with age. Indole glucosinolate content was identical in both cauliflower and broccoli, and both indole and aromatic glucosinolates did not vary with plant age in either crop. Qualitative differences in characterized glucosinolates were observed between broccoli and cauliflower but no differences were observed between inoculated and noninoculated plants for either broccoli or cauliflower. However, the phenolic and lignin contents were significantly higher in broccoli following inoculation than in noninoculated broccoli or inoculated cauliflower plants. The increased resistance of broccoli to V. dahliae infection was related to the increase in phenolic and lignin contents. Significant differential accumulation of glucosinolates associated with plant phenology may also contribute to the resistant and susceptible reactions of broccoli and cauliflower, respectively, against V. dahliae.

Interactions between Coniothyrium minitans and Sclerotinia minor affect biocontrol efficacy of C. minitans.

Coniothyrium minitans, marketed as Contans, has become a standard management tool against Sclerotinia sclerotiorum in a variety of crops, including winter lettuce. However, it has been ineffective against lettuce drop caused by S. minor. The interactions between C. minitans and S minor were investigated to determine the most susceptible stage in culture to attack by C. minitans, and to determine its consistency on S minor isolates belonging to four major mycelial compatibility groups (MCGs). Four isolates of S. minor MCG 1 and 5 each from MCGs 2 and 3 and one from MCG 4 were treated in culture at purely mycelial, a few immature sclerotial, and fully mature sclerotial phases with a conidial suspension of C. minitans. Sclerotia from all treatments were harvested after 4 weeks, air dried, weighed, and plated on potato dextrose agar for recovery of C. minitans. S. minor formed the fewest sclerotia in plates that received C. minitans at the mycelial stage; C. minitans was recovered from nearly all sclerotia from this treatment and sclerotial mortality was total. However, the response of MCGs was inconsistent and variable. Field experiments to determine the efficacy of C. minitans relative to the registered fungicide, Endura, on lettuce drop incidence and soil inoculum dynamics were conducted from 2006 to 2009. All Contans treatments had significantly lower numbers of sclerotia than Endura and unsprayed control treatments, and drop incidence was as low as in Endura-treated plots (P > 0.05). Although the lower levels of lettuce drop in Contans treatments were correlated with significantly lower levels of sclerotia, the lower levels of lettuce drop, despite the presence of higher inoculum in the Endura treatment, was attributable to the prevention of infection by S. minor. A useful approach to sustained lettuce drop management is to employ Contans to lower the number of sclerotia in soil and to apply Endura to prevent S. minor infection within a cropping season.