First Report of Gliocephalotrichum bulbilium Causing Cranberry Fruit Rot in New Jersey and Massachusetts.

Cranberry (Vaccinium macrocarpon) fruit were collected as part of a fruit rot survey conducted in September 2010 on farms in New Jersey and Massachusetts. There are more than 20 fungal species reported as causing fruit rot (2) and symptoms are generally not diagnostic. The rotted fruit were surface sterilized in a 10% bleach solution for 5 min, sliced in half, and plated on V8 agar (nonclarified). A novel, fast-growing fungus that produced sporulating orange-brown colonies emerged from 5% of the fruit collected on three of the farms included in the survey. The fungus was notable as the only species present in the rotted fruit, suggesting it may be pathogenic. The conidia were produced as gloeoid masses on phialidic conidiogenous cells arranged in a polyverticillate penicillus. The conidiogenous cells were subtended at variable distances by zero to four sterile appendages that formed on the lightly pigmented conidiophore. On the basis of these characteristics, the fungus was identified as a species of Gliocephalotrichum (3). Further investigation of the growth medium revealed the presence of clustered, red-brown chlamydospores that were produced abundantly in all isolates. These structures, also known as bulbils, are restricted to two species in the genus, G. bulbilium and G. longibrachium (1). On average, the bulbils were 42.0 × 48.3 µm and conidia were 5.75 × 2.5 µm. On the basis of size and shape of conidia and presence of bulbils, the isolates were identified as G. bulbilium (1). To confirm the identity of the fungus, genomic DNA was extracted and ITS1-5.8S-ITS2 and the 5' end of the ß-tubulin gene were amplified and sequenced (1). The sequences (GenBank Accession Nos. HQ828060 and HQ828061) were compared with published sequences of Gliocephalotrichum isolates (1) and results confirmed the cranberry isolates were G. bulbilium. The isolates were tested for pathogenicity on harvested cranberry fruit. Fifty ripe cranberry fruit (cv. Stevens) were inoculated by injecting approximately 20 µl (using a 26G 9.5-mm needle) of conidia (1 × 105 ml-1) into the side of each berry. As a comparison, isolates of two common cranberry fruit rot pathogens, Colletotrichum acutatum and C. gloeosporioides, were inoculated on to fruit using the same technique. A water-only inoculation was used as the control. Fruit rot developed on all inoculated fruit except the water control. In the case of G. bulbilium, all fruit rotted within 2 days, whereas the other two species developed symptoms within 4 to 7 days. G. bulbilium and both species of Colletotrichum were consistently reisolated from all of the respectively inoculated fruit. To our knowledge, this is the first report of G. bulbilium causing fruit rot on cranberry. The species has been reported as an important postharvest fruit rot (4) on rambutan (Nephelium lappaceum) in Thailand, rambutan and guava (Psidium guajava) in Hawaii, and durian (Durio spp.) in Brunei Darussalam. This report of G. bulbilium extends the range within the United States to include Louisiana, Hawaii, Wisconsin, West Virginia, New Jersey, and Massachusetts (2). References: (1) C. Decock et al. Mycologia 98:488, 2006. (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/ , 16 December 2010. (3) A. Rossman et al. Mycologia, 85:685, 1993. (4) A. Sivapalan et al. Australas. Plant Pathol. 27:274, 1998.

First Report of Blueberry scorch virus on Highbush Blueberry in Connecticut and Massachusetts.

During June and July 2001, the Marucci Center received 33 foliage samples from healthy- and unhealthy-looking highbush blueberry (Vaccinium corymbosum L.) bushes from growers in Connecticut and Massachusetts, via local extension agents. Unhealthy bushes were reported to exhibit symptoms including leaf chlorosis and necrosis, blossom blight, tip dieback, or a general decline in vigor. Marginal leaf chlorosis, reddening, or necrosis characterized foliage samples from these bushes. Five-leaf samples from each bush were tested for Blueberry scorch virus (BlScV) (3) with Agri-Check detection kits (Hydros, Inc., Falmouth, MA). These kits use an indirect enzyme-linked immunosorbent assay (ELISA) protocol and antibodies developed at Rutgers University that are specific to the two eastern strains of BlScV (NJ1 and NJ2) (1). The ELISA extraction buffer was based on that used by Martin and Bristow (3) with 1% (wt/vol) nonfat dry milk powder added as a blocking agent. Fourteen samples from cvs. Blueray and Berkeley in both states and cvs. Elliott, Bluecrop, and Coville in Massachusetts tested positive for BlScV. These results were confirmed by a second test. Six of seven samples from symptomatic bushes and 8 of 26 samples from asymptomatic bushes harbored BlScV. Virus preparations extracted from five infected plants (two from Connecticut and three from Massachusetts) were examined using reverse transcription-polymerase chain reaction (RT-PCR) with oligonucleotide primers (5'-TGTGTCAAACAATATGGC-3' and 5'-GCATTTCGATGA-TTGCGG-3') designed to amplify a portion of the coat protein gene from any of the three known virus strains (1,2). Sequence analysis of fragments amplified from their coat protein genes revealed that two of the isolates from Massachusetts (GenBank Accession Nos. AY530957 and AY530958) and the two isolates from Connecticut (GenBank Accession Nos. AY530955 and AY530956) were similar but not identical to one another, and these four were most similar to strain NJ2. One isolate from Massachusetts (GenBank Accession No. AY530958) was most similar to strain NJ1. To our knowledge, this is the first report of BlScV on the east coast outside of New Jersey, where it was first reported in 1983 (4). These results indicate that the disease is now present in other blueberry-growing areas in the northeast and is likely to be spreading locally within those areas. Because blueberry scorch is symptomless in propagation material and may take several years to express symptoms in the field, the initial spread of the disease was probably due to the shipping of latently infected plants to BlScV-free areas before reliable testing was available. References: (1) T. D. Cavileer et al. J. Gen. Virol. 75:711, 1994. (2) B. T. Halpern and B. I. Hillman. Plant Dis. 80:219, 1996. (3) R. R. Martin and P. R. Bristow. Phytopathology 78:1636, 1988. (4) A. W. Stretch. (Abstr.) Phytopathology 73:375, 1983.

Variation and Heritability of Phenology in the Fungus Monilinia vaccinii-corymbosi on Blueberry.

ABSTRACT The germination of field-collected pseudosclerotia and the development of apothecia from eight New Jersey populations of the mummy berry fungus Monilinia vaccinii-corymbosi were evaluated under controlled conditions in the greenhouse. Development data for apothecia were used to describe the timing of apothecium formation and to estimate broad- and narrow-sense heritabilities of fungal phenology. Mean development times for the formation of apothecia ranged from 35.4 to 54.7 days. The mean development times for populations collected from early-season cv. Weymouth ranged from 35.4 to 39.6 days and were significantly shorter than the development times for three of the four populations collected from late-season cv. Jersey (46.9 to 54.7 days) or for the population collected from mixed stands of cultivated blueberries (42.7 days). The development of populations from late cultivars planted in very close proximity to early cv. Weymouth was early (36.5 to 39.0 days) and not significantly different from the development of populations collected from cv. Weymouth. Phenotypic and genetic variances of apothecium development for individual populations ranged from 18.9 to 44.8 and 7.2 to 30.9, respectively. Broad-sense heritabilities of apothecia development for each fungal population, calculated by partitioning phenotypic variation into genetic and environmental components, ranged from 0.31 to 0.78. Narrow-sense heritabilities of apothecia development, based on parent-offspring regression, ranged from 0.58 to 0.78. These results indicate that populations of M. vaccinii-corymbosi differ in phenology and that a significant portion of the phenological variation within populations is genetic. Thus, it is plausible to propose that the phenology of apothecium development is a component of fungal fitness and that host phenology can influence the timing of pathogen development.

A long terminal repeat retrotransposon Cgret from the phytopathogenic fungus Colletotrichum gloeosporioides on cranberry.

A repetitive DNA element cloned from the cranberry fruit rot pathogen Colletotrichum gloeosporioides has been characterized. Sequence data indicate that it is a long terminal repeat (LTR) retrotransposon of 7,916 base pairs. LTR of 544 base pairs occur at either end of an internal region of 6,828 base pairs. This element, designated Cgret (C. gloeosporioides retrotransposon), encodes two putative polypeptides which have high homology to the gag and pol genes of other fungal retrotransposons. The sequence and structure suggest that Cgret is a member of the gypsy group of LTR retrotransposons. The Cgret retrotransposon was present in all of the cranberry isolates of the fungus C. gloeosporioides from New Jersey and Massachusetts, but not in the cranberry isolates from Wisconsin or Chile. Polymorphisms were detected among field isolates of C. gloeosporioides from various hosts, using hybridization probes derived from the LTR and the reverse transcriptase domain of Cgret. The structural integrity of Cgret suggests that it is still a functional retrotransposon and may be used as a molecular marker to study the genetic diversity distribution of this fungal pathogen.

Distribution of cranberry fruit-rotting fungi in new jersey and evidence for nonspecific host resistance.

ABSTRACT A survey was conducted over a 3-year period to determine the frequencies and distributions of fruit-rotting fungi in New Jersey cranberry beds. In the first 2 years of the study, Physalospora vaccinii and Glomerella cingulata were the most prevalent and widespread field-rotting fungi. In the third year, the frequency of G. cingulata declined markedly. Other species such as Coleophoma empetri, Phyllosticta vaccinii, and Phomopsis vaccinii were isolated at high frequencies from a limited number of locations. Storage-rotting fungi including Allantophomopsis cytisporea and A. lycopodina were isolated at low frequencies, but were widely distributed within the growing region. On sound fruit, a somewhat different profile emerged. Fungi such as Phyllosticta elongata, Alternaria spp., and Physalospora vaccinii were commonly isolated. In comparisons among different cranberry cultivars, no differences in the fungal profiles were seen. This was interpreted to indicate that if differences in fruit-rot resistance exist, they are likely to be general forms of resistance rather than fungal species-specific mechanisms.

Phenology of Apothecium Production in Populations of Monilinia vaccinii-corymbosi from Early- and Late-Maturing Blueberry Cultivars.

ABSTRACT Pseudosclerotia were evaluated for differences in timing of apothecium development in four controlled experiments conducted over a 2-year period. In a separate experiment, conidia from 10 randomly selected isolates from both of the fungal populations were used to inoculate open flowers. Germination of pseudosclerotia produced from these artificial inoculations also was evaluated. The timing and rate of shoot elongation for cvs. Weymouth and Jersey were assessed in one greenhouse and two field experiments. Average development times for the fungal population from cv. Weymouth were 8 to 15 days earlier or 33 to 42% less than those for the population from cv. Jersey. The fungal population from Weymouth also exhibited less variation in development times for each developmental stage measured. Similarly, germination of pseudosclerotia produced in artificial inoculations differed between populations. On average, pseudosclerotia derived from the Weymouth population produced apothecia 16 days earlier. During spring 1995 and 1996, vegetative and truss buds on cv. Weymouth developed 4 to 16 days earlier than those on cv. Jersey. These results demonstrate that M. vaccinii-corymbosi exhibits variation in timing of pseudosclerotia germination and apothecium development within and between populations. We hypothesize that differences observed in the timing of apothecium development are related to the fitness of the populations on their original host cultivars and were selected by host phenology.