Sensitivity and Efficacy of Boscalid, Fluazinam, and Thiophanate-Methyl for White Mold Control in Snap Bean in New York.

White mold (Sclerotinia sclerotiorum) of leguminous crops in New York is generally managed with preventive applications of fungicides. However, no research has been conducted during the last decade to assess the sensitivity of the S. sclerotiorum population to fungicides or compare their performance under field conditions. The sensitivity of S. sclerotiorum to boscalid, fluazinam, and thiophanate-methyl was assessed in 151 isolates from 15 fields across New York using an agar dilution method with discriminatory concentrations. In addition, the effective concentration at which mycelial growth is reduced by 50% (EC50) was estimated for one representative isolate from each field. The efficacy of commercial formulations of each fungicide on white mold incidence in plants and pods was also tested in two field trials (2015 and 2016). The EC50 values ranged from 0.068 to 0.219, 0.001 to 0.002, and 1.23 to 2.15 µg/ml for boscalid, fluazinam, and thiophanate-methyl, respectively. Evidence of resistance was not found using the discriminatory concentration tests. The mycelial growth inhibition relative to the control ranged from 56 to 83%, 66 to 84%, and 53 to 83% at discriminatory concentrations of boscalid (5 µg a.i./ml), fluazinam (0.05 µg a.i./ml), and thiophanate-methyl (5 µg a.i./ml), respectively. Fourteen isolates with mycelial growth inhibition lower than 60% at 5 µg/ml of thiophanate-methyl, did not exhibit point mutations within a partial sequence of the ß-tubulin gene. In the field trials, fungicides effectively reduced white mold incidence on plants by 75% (2015) and 93% (2016) and on pods by 81% (2015) and 87% (2016), both relative to the nontreated plots. However, fungicide applications led to significant increases in pod yield, relative to the nontreated plots, only in 2015 when the incidence of white mold on plants and pods were higher (85 and 49.2%) than in 2016 (31.3 and 10.3%). Although fungicide resistance was not detected, and thus control failures reported by New York snap bean growers may be due to other factors, further monitoring of sensitivity within the S. sclerotiorum population is encouraged as well as the use of rational systems to base their judicious and economic use.

First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia.

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite 'E' (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 µm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite 'E'. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5'-AGTGCGGTGGTATCGACAAG) and EFCF6 (3'-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of a 16SrII-D Subgroup Phytoplasma Associated with Pale Purple Coneflower Witches'-Broom Disease in Australia.

Pale purple coneflower, Echinacea pallida (Nutt.) Nutt., is an herbaceous perennial cultivated for its ornamental and medicinal properties. In 2005, phytoplasma-like symptoms, including virescence, phyllody, and chlorotic leaves, were first observed in coneflower fields in northern Tasmania, Australia. During the 2010-2011 growing season, the incidence of affected plants was estimated to be 12% within a single field. Total DNA was extracted from symptomatic plants with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions. DNA was also extracted, as described above, from asymptomatic coneflower seedlings obtained by germinating surface-sterilized seed on water agar. DNA was amplified via a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 to detect putative phytoplasmas (2). Amplifications yielded expected products of 1.8 and 1.2 kb, respectively, only from symptomatic samples. Subsequently, PCR products from six arbitrarily selected samples were used for sequencing (Genome Lab Dye Terminator Cycling Sequence with Quick Start Chemistry) and read in a CEQ8000 sequencer (Beckman Coulter Inc., Brea, CA). Sequence homology indicated 100% similarity between isolates designated EWB1 to EWB4 (GenBank Accession Nos. JF340075 and JF340077 to JF340079) and between EWB5 and EWB6 (JF340076 and JF40080). Sequence homology between the two observed groups was 99.7%, resulting from a 4-bp difference in the R16F2n primer region. Blast search revealed isolates EWB1 to EWB4 were 100% homologous with Catharanthus roseus phytoplasma (EU096500.1), Tomato big bud phytoplasma (EF193359.1), Scaevola witches'-broom phytoplasma (AB257291.1), and Mollicutes sp. (Y10097.1 and Y10096.1). Moreover, isolates EWB5 and EWB6 shared 99% sequence identity with the above isolates. iPhyClassifier (4) was used to perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of isolates EWB1 to EWB4 and EWB5 to EWB6 shared 100 and 99.7% similarity, respectively, to the 'Candidatus Phytoplasma australasiae' reference strain (Y10097). RFLP profiles from all isolates suggested that they belonged to the 16SrII-D subgroup. To our knowledge, this is the first report of a 16SrII-D subgroup phytoplasma infecting E. pallida in Australia. Aster yellow phytoplasmas (16SrI-C subgroup) infections of E. purpurea have been recorded in Slovenia (3) and southern Bohemia (1). References: (1) J. Franova et al. Eur. J. Plant Pathol. 123:85, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) S. Radisek et al. Plant Pathol. 58:392, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

First Report of Fusarium crookwellense Causing Tip Blight on Cones of Hop.

Hop (Humulus lupulus L.) is grown primarily for the alpha and beta acids produced in the strobile (cone) and used for bittering beer. In late summer (March) 2001, necrotic lesions covering the tips of cones of cvs. Agate, Nugget, and Willamette at hop farms in Tasmania, Australia, were observed. The necrotic lesions encompassed the proximal tips and affected between 5 and 60% of the cone; however, all bracts in the whorl were always affected. Diseased cones were observed in all seven gardens included in the survey. The incidence of plants with cone tip blight in 'Nugget' ranged from 5 to 30% in three gardens, in 'Agate' ranged from 3 to 10% in three gardens, and in the only 'Willamette' garden 30% of cones were affected. Pieces of infected hop cones (N = 55) were surface-treated for 1 min in 2% sodium hypochlorite, placed on 2% water agar, and incubated at 22 ± 2°C. Fusarium crookwellense Burgess, Nelson, & Toussoun was isolated from 95% of the cones (1). F. crookwellense was identified on carnation leaf agar by L. Burgess, University of Sydney, Australia. Koch's postulates were fulfilled by inoculating detached mature hop cones of cvs. Nugget and Willamette (N = 20 for each cultivar) with an atomized conidial suspension (3.5 × 105 spores of a single F. crookwellense isolate per milliliter) until runoff and incubated at 20 ± 2°C in a sealed container on plastic mesh over tissue wetted with sterile distilled water. Symptoms first appeared 5 days after inoculation and were identical to those found in the field. No disease symptoms were observed on cones subjected only to sterile distilled water. The pathogen was reisolated from diseased tissue on inoculated cones, completing Koch's postulates. Similar disease symptoms on hop cones have been described in Oregon and were associated with infection by F. sambucinum and F. avenaceum (C. Ocamb, personal communication). To our knowledge, this is the first report of the infection of hop cones by F. crookwellense. Reference: (1) L. W. Burgess et al. Laboratory Manual for Fusarium Research, 3rd ed. University of Sydney, Australia, 1994.

First Report of Infection of Hop Cones by Alternaria alternata in Australia.

The hop (Humulus lupulus L.) is a dioecious climbing plant, cultivated for its resins, which are produced in the cone, used primarily for the bittering of beer. In Australia, hops are grown in the states of Victoria and Tasmania. In late summer 2001, necrotic lesions were observed on the tips of bracts and bracteoles of developing cones at three hop farms in Tasmania. The necrotic area varied between 1 and 25% of the bracts and bracteoles, and in some cases progressed throughout the entire hop cone. Pieces of infected hop cones were surface sterilized for one minute in 2% sodium hypochlorite, plated on 2% water agar, and incubated at 22 ± 2°C. The most frequently isolated fungi (total number of isolations = 60) were transferred to 2% water agar and potato dextrose agar. In 90% of cases, the isolated fungus was Alternaria alternata (Fr.:Fr.) Keissl, as identified by M. Priest, NSW Agriculture, Australia. The pathogenicity of A. alternata was determined on detached, freshly picked cones of hop cultivar "Nugget." Cones (n = 25) were inoculated with a conidial suspension of the fungus (1,000 spores per ml) and incubated at room temperature in a sealed container on plastic mesh over tissue wetted with sterile distilled water. Symptoms first appeared three days after inoculation as necrotic tips of bracts and bracteoles, and within 10 days the entire cone had become necrotic. Symptoms were more severe in vitro compared to in the field. This was probably due to the maintenance of detached cones under constant high relative humidity. Disease symptoms did not appear on cones inoculated with sterile distilled water. The pathogen was reisolated from inoculated cones, completing Koch's postulates. The pathogenicity of A. alternata to hop cones was reported in the United Kingdom in 1988. To our knowledge, this is the first report of A. alternata on hop cones in Australia. References: (1) P. Darby. Trans. Br. Mycol. Soc. 90:650-653, 1988.

First Report of Tomato spotted wilt virus in Common Agapanthus.

Common agapanthus (Agapanthus praecox subsp. orientalis), native to South Africa, is a popular ornamental flowering bulb species belonging to the Amaryllidaceae and is commonly found in residential gardens. Roots from some Agapanthus sp. also are used in traditional medicine in Africa. Common agapanthus collected from a residential property in Hobart, Tasmania, Australia, showed leaf symptoms of concentric ring and line patterns, irregular chlorotic blotches, and streaks. Symptomatic plants were severely stunted and failed to flower. Symptomatic leaves prematurely senesced, but young foliage subsequently produced was symptomless. Similar symptoms have been reported in other members of the Amaryllidaceae and are associated with infection by Tomato spotted wilt virus (TSWV; e.g., Nerine and Hippeastrum spp.) or Cucumber mosaic virus (CMV; e.g., Hippeastrum sp.) (2). The presence of TSWV and absence of CMV in symptomatic plants of common agapanthus was determined by enzyme-linked immunosorbent assay. Confirmation of TSWV infection was provided by reverse-transcription polymerase chain reaction assay with primers specific to the nucleocapsid protein gene of TSWV, with nucleic extracts from symptomatic plants producing an expected ≈800-bp amplicon (1). This is the first report of TSWV infection of any species within the Amaryllidaceae in Australia and the first report of the occurrence of TSWV in common agapanthus. References: (1) R. K. Jain et al. Plant Dis. 82:900, 1998. (2) G. Loebenstein et al. 1995. Virus and Virus-like Diseases of Bulb and Flower Crops. John Wiley & Sons, Chichester, U.K.