New Species-Specific Real-Time PCR Assays for Colletotrichum Species Causing Bitter Rot of Apple.

Bitter rot of apple is an economically important worldwide disease caused by different Colletotrichum species, depending on many factors such as climate, geography, other hosts, and crop management practices. Culture, morphology, and single-locus sequencing-based methods for identifying the Colletotrichum species are severely limited in effectiveness, while the multilocus sequence typing methods available for delineating species are costly, time-intensive, and require high expertise. We developed species-specific hydrolysis probe real-time PCR assays for the following nine Colletotrichum species causing bitter rot in the Mid-Atlantic U.S.A.: C. fructicola, C. chrysophilum, C. noveboracense, C. gloeosporioides s.s., C. henanense, C. siamense and C. theobromicola from the C. gloeosporioides species complex, and C. fioriniae and C. nymphaeae from the C. acutatum species complex. After searching 14 gene regions, we designed primers and probes in 5 of them for the nine target species. Four primer-probe set pairs were able to be duplexed. Sensitivity tests showed as little as 0.5 pg DNA were detectable. These real-time PCR assays will provide rapid and reliable identification of these key Colletotrichum species and will be critically important for studies aiming to elucidate their biology, epidemiology, and management on apples as the number one produced and consumed tree fruit in the U.S.A.

A Bitter, Complex Problem: Causal Colletotrichum Species in Virginia Orchards and Apple Fruit Susceptibility.

Bitter rot, caused by Colletotrichum species, is one of the most devastating summer rot diseases affecting apple production in the Eastern United States. Given the differences in virulence and fungicide sensitivity levels between organisms belonging to the acutatum species complex (CASC) and the gloeosporioides species complex (CGSC), monitoring their diversity, geographic distribution, and frequency are essential for successful bitter rot management. In a 662-isolate collection from apple orchards in Virginia, isolates from CGSC were dominant (65.5%) in comparison to the CASC (34.5%). In a subsample of 82 representative isolates, using morphological and multilocus phylogenetic analyses, we identified C. fructicola (26.2%), C. chrysophilum (15.6%), C. siamense (0.8%), and C. theobromicola (0.8%) from CGSC and C. fioriniae (22.1%) and C. nymphaeae (1.6%) from CASC. The dominant species were C. fructicola, followed by C. fioriniae and C. chrysophilum. C. siamense followed by C. theobromicola developed the largest and deepest rot lesions on Honeycrisp fruit in our virulence tests. Detached fruit of nine apple cultivars and one wild accession (Malus sylvestris) were harvested early and late season and tested in controlled conditions for their susceptibility to C. fioriniae and C. chrysophilum. All cultivars were susceptible to both representative bitter rot species, with Honeycrisp fruit being the most susceptible and M. sylvestris, accession PI 369855, being the most resistant. We demonstrate that the frequency and prevalence of species in Colletotrichum complexes are highly variable in the Mid-Atlantic and provide region-specific data on apple cultivar susceptibility. Our findings are necessary for the successful management of bitter rot as an emerging and persistent problem in apple production both pre- and postharvest.

Jikradia olitoria ([Hemiptera]:[Cicadellidae]) Transmits the Sequevar NAGYIIIß Phytoplasma Strain Associated with North American Grapevine Yellows in Artificial Feeding Assays.

North American Grapevine Yellows (NAGY) is a destructive disease of grapevines caused by phytoplasmas, wall-less bacteria that are insect-transmitted and found in plant phloem tissues. Although the disease was recognized in vineyards in the eastern United States since the 1980s, the identities of vectors remain unknown. The objectives of this study were to survey potential phytoplasma vector insects inhabiting Virginia vineyards that expressed NAGY symptoms and to evaluate their ability to transmit phytoplasmas associated with NAGY. Phytoplasmas were identified as 'Candidatus Phytoplasma pruni'-related NAGYIIIß strains and 'Ca. Phytoplasma asteris'-related NAGYI-B strains. To determine the identities of the potential vectors, artificial feeding solution was used to evaluate the ability of leafhopper species to release phytoplasmas during feeding and phytoplasma strains were identified using molecular tools. Out of 49 insect species screened, Jikradia olitoria was the only insect that released phytoplasmas into the feeding solutions; all phytoplasmas, thus, detected were identified as NAGYIIIß strains by nucleotide sequencing of three different genomic regions. No NAGYI-B strain was detected. To our knowledge, this is the first evidence of a potential insect vector of a specific phytoplasma associated with NAGY disease, and it is the first report of J. olitoria being a putative vector of a plant pathogenic phytoplasma.

Co-infection of blacklegged ticks with Babesia microti and Borrelia burgdorferi is higher than expected and acquired from small mammal hosts.

Humans in the northeastern and midwestern United States are at increasing risk of acquiring tickborne diseases--not only Lyme disease, but also two emerging diseases, human granulocytic anaplasmosis and human babesiosis. Co-infection with two or more of these pathogens can increase the severity of health impacts. The risk of co-infection is intensified by the ecology of these three diseases because all three pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti) are transmitted by the same vector, blacklegged ticks (Ixodes scapularis), and are carried by many of the same reservoir hosts. The risk of exposure to multiple pathogens from a single tick bite and the sources of co-infected ticks are not well understood. In this study, we quantify the risk of co-infection by measuring infection prevalence in 4,368 questing nymphs throughout an endemic region for all three diseases (Dutchess County, NY) to determine if co-infections occur at frequencies other than predicted by independent assortment of pathogens. Further, we identify sources of co-infection by quantifying rates of co-infection on 3,275 larval ticks fed on known hosts. We find significant deviations of levels of co-infection in questing nymphs, most notably 83% more co-infection with Babesia microti and Borrelia burgdorferi than predicted by chance alone. Further, this pattern of increased co-infection was observed in larval ticks that fed on small mammal hosts, but not on meso-mammal, sciurid, or avian hosts. Co-infections involving A. phagocytophilum were less common, and fewer co-infections of A. phagocytophilum and B. microti than predicted by chance were observed in both questing nymphs and larvae fed on small mammals. Medical practitioners should be aware of the elevated risk of B. microti/B. burgdorferi co-infection.

Prevalence of human-active and variant 1 strains of the tick-borne pathogen Anaplasma phagocytophilum in hosts and forests of eastern North America.

Anaplasmosis is an emerging infectious disease caused by infection with the bacterium Anaplasma phagocytophilum. In the eastern United States, A. phagocytophilum is transmitted to hosts through the bite of the blacklegged tick, Ixodes scapularis. We determined the realized reservoir competence of 14 species of common vertebrate hosts for ticks by establishing the probability that each species transmits two important strains of A. phagocytophilum (A. phagocytophilum human-active, which causes human cases, and A. phagocytophilum variant 1, which does not) to feeding larval ticks. We also sampled questing nymphal ticks from ∼ 150 sites in a single county over 2 years and sampled over 6 years at one location. White-footed mice (Peromyscus leucopus) and Eastern chipmunks (Tamias striatus) were the most competent reservoirs for infection with the A. phagocytophilum human-active strain. Across the county, prevalence in ticks for both strains together was 8.3%; ticks were more than two times as likely to be infected with A. phagocytophilum human-active as A. phagocytophilum variant 1.

Reservoir competence of vertebrate hosts for Anaplasma phagocytophilum.

Fourteen vertebrate species (10 mammals and 4 birds) were assessed for their ability to transmit Anaplasma phagocytophilum, the bacterium that causes human granulocytic anaplasmosis, to uninfected feeding ixodid ticks. Small mammals were most likely to infect ticks but all species assessed were capable of transmitting the bacterium, in contrast to previous findings.