F. S. Hay

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. Kochs 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 Kochs 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 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 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 Kochs 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.

Isolation of nematode DNA from 100 g of soil using Fe 3 O 4 super paramagnetic nanoparticles

An economical method for extracting nematode DNA from 100xa0g of soil was developed to facilitate nematode detection and quantification, and tested using the Northern root-knot nematode, Meloidogyne hapla . The method utilised enzymatic laundry detergent lysis, Fe 3 O 4 super paramagnetic iron oxide nanoparticle (SPION) capture, and polyvinylpolypyrrolidone (PVPP) purification. Resultant DNA from this SPION capture method was approximately 100-fold less but of similar quality to DNA obtained from a standard phenol procedure and a commercial DNA extraction kit. An addition of 10xa0mg of nanoparticles to the extraction lysate was identified to maximise DNA yield while minimising co-capture of contaminants. The detection limit of the SPION capture method was approximately 100 nematodes (100xa0g soil) −1 . The SPION capture method extracted nematode DNA from mineral soils but requires further optimisation for extraction from high organic matter ( i.e. , ‘muck’) soils. The benefits of this method compared to alternative techniques are discussed.

Identification and characterization of Ditylenchus spp. populations from garlic in New York State, USA

Ditylenchus dipsaci is an important pathogen of onion and garlic and causes reductions in bulb quality. Between 2012 and 2014, 345 garlic bulb samples were tested for Ditylenchus spp. The average incidence of Ditylenchus spp. was 14.1xa0%. Twenty-five individuals from 31 Ditylenchus spp. populations were selected for identification by morphology, species-specific PCR, and sequencing of the ITS1-5.8S-ITS2 region. Thirty populations were identified by morphology and species-specific PCR as D. dipsaci. Sequencing of the ITS1-5.8S-ITS2 region of the ribosomal DNA found the populations were 99.7 to 100xa0% similar to each other, and 99.3 to 100xa0% similar to other D. dipsaci reference isolates from France, and California, USA. One population was identified as Ditylenchus sp. and was 97xa0% similar within the ITS1-5.8S-ITS2 region to D. destructor. This information will be useful for the monitoring of future pest outbreaks in garlic in New York State, USA, and the effectiveness of management recommendations.

Revisiting Stagonosporopsis species associated with chrysanthemum and pyrethrum ray blight

Ray blight is a destructive disease of Asteraceae affecting chrysanthemum and pyrethrum industries worldwide. Three morphologically similar but phylogenetically distinct species of the family Didymellaceae; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti are associated with the disease. Despite their close evolutionary relationship and cross host pathogenicity, these species have marked differences in their biology and epidemiology. Stagonosporopsis chrysanthemi and S. inoxydabilis are both homothallic with a MAT locus that carries both mating type genes. Ascomata play a major role in survival and dispersal of S. chrysanthemi, contributing to the onset of ray blight epidemics on chrysanthemum. However, S. tanaceti is either asexual or heterothallic due to the presence of only MAT1-2 idiomorph in its genome. Morphological similarity of the species causing ray blight on various Asteraceae, multiple changes in their taxonomy and historical confusion with other Phoma-like species have resulted in lack of clarity in their taxonomy, host range and global distribution. Host specificity studies and reports on global distribution of S. chrysanthemi published before its separation from S. inoxydabilis should be treated with caution. A recently developed species-specific multiplex PCR assay provides a rapid and robust tool to study the global distribution of these important quarantine pathogens. When global populations from cultivated and/or wild Asteraceae are available, population genetic analyses may aid in understanding the origin, evolutionary history and global migration patterns of the Stagonosporopsis spp. associated with ray blight of Asteraceae.

Cryptic diversity, pathogenicity, and evolutionary species boundaries in Cercospora populations associated with Cercospora leaf spot of Beta vulgaris

The taxonomy and evolutionary species boundaries in a global collection of Cercospora isolates from Beta vulgaris was investigated based on sequences of six loci. Species boundaries were assessed using concatenated multi-locus phylogenies, Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Processes (PTP), and Bayes factor delimitation (BFD) framework. Cercospora beticola was confirmed as the primary cause of Cercospora leaf spot (CLS) on B.xa0vulgaris. Cercospora apii, C. cf. flagellaris, Cercospora sp. G, and C.xa0zebrina were also identified in association with CLS on B. vulgaris. Cercospora apii and C. cf. flagellaris were pathogenic to table beet but Cercospora sp. G and C.xa0zebrina did not cause disease. Genealogical concordance phylogenetic species recognition, GMYC and PTP methods failed to differentiate C.xa0apii and C.xa0beticola as separate species. On the other hand, multi-species coalescent analysis based on BFD supported separation of C.xa0apii and C.xa0beticola into distinct species; and provided evidence of evolutionary independent lineages within C.xa0beticola. Extensive intra- and intergenic recombination, incomplete lineage sorting and dominance of clonal reproduction complicate evolutionary species recognition in the genus Cercospora. The results warrant morphological and phylogenetic studies to disentangle cryptic speciation within C.xa0beticola.

First Report of Cercospora Blight of Asparagus officinalis Caused by Cercospora asparagi in New York

In October 2016, disease was observed on stems and branches of asparagus (Asparagus officinalis) in an organic crop at a fresh market farm in Ontario Co., NY. Lesions were small (< 2 to 3 cm in length), generally oval and gray to tan with a reddish-brown margin. The disease was associated with defoliation on approximately 40% of plants. Conidiophores and conidia typical of Cercospora were observed within the lesions. Morphological characteristics of the fungus were consistent with descriptions of C. asparagi (Braun et al. 2014). Conidiomata were brown, consisting of dense conidiophores on a defined stroma (25 to 100 µm long × 2.5 to 4.5 µm wide). Conidia were solitary, hyaline, straight to curved, acicular, with hila at the base (40 to 90 µm long × 2.5 to 4 µm wide). Fifty µl of T-water (0.06% v/v Tween-20 + 0.02% w/v ampicillin) were placed on individual sporulating lesions. The conidial suspension was transferred to water agar (+ 0.02% w/v ampicillin) and incubated at room temperature. Cultures arising ...

First report of carrot root rot caused by Rhexocercosporidium carotae in the United States.

In March 2015, black irregularly shaped lesions up to 30 mm in diameter were observed on carrot roots that were placed in cold storage for 6 months in Wadhams, Essex County, New York. Disease incidence was ∼5% and up to 30% of the tissue was affected on some roots.