Spruce Decline and Diaporthe: Incidence, Taxonomy, Virulence, and Tree Susceptibility in Michigan.

In the early 2000s, spruce trees in Michigan began displaying basal needle drop and branch death that slowly progressed upward, symptoms of what we call spruce decline. A survey in 2013 revealed that spruce decline was common throughout Michigan's Lower Peninsula, and Diaporthe was the most likely pathogen causing the cankers associated with these symptoms. Greenhouse inoculation studies completed Koch's postulates, confirming that Diaporthe could cause cankers that cause needle loss and branch death. The five different Diaporthe haplotypes isolated from symptomatic branches during the survey differed in virulence. Haplotypes 2, 4, and 5 were more virulent, and differed from each other by only one or two base pairs using the internal transcribed spacer (ITS) region and did not differ using the ß-tubulin (TUB) gene. These haplotypes were unresolved phylogenetically. Haplotypes 1 and 3 were weakly virulent to avirulent on multiple spruce taxa, and fell into a resolved Diaporthe eres clade. Spruce taxa varied in susceptibility, with Colorado blue spruce (Picea pungens) the most susceptible, followed by Norway (P. abies), then white spruce (P. glauca). Spruce taxa that were much less susceptible were Black Hills (P. glauca var. densata), Serbian (P. omorika), and Meyer spruce (P. meyeri). We demonstrate that one or more Diaporthe species is causing cankers on declining spruce in Michigan, and these cankers elicit symptoms similar to the branch death expressed by declining spruce in the landscape. Future work will focus on further characterizing Diaporthe to species, and determining biotic and abiotic stressors that may predispose spruce trees to express decline symptoms.

First Report of a 16SrIX Group (Pigeon Pea Witches'-Broom) Phytoplasma Associated with Sesame Phyllody in Turkey.

Sesame (Sesamum indicum L.) is an important oilseed crops widely grown in the southern regions of Turkey. Sesame seeds are primarily used in production of tahini as well as a garnish on sweets and bakery products in the country. Sesame plants with phyllody disease symptoms have increasingly been observed in the fields of Antalya province since 2007. The disease incidence in these fields was found to range from 37 to 62% (2). Infected plants display a variety of the disease symptoms such as virescence, asymptomatic shoot proliferation, infertile flower formation, reduced leaf size, and thin and weak capsule development. Total genomic DNA was extracted from samples collected from symptomatic (10 plants) and asymptomatic healthy-looking plants (10 plants) using a CTAB method and amplified with universal primers P1/P7 and R16F2n/R16R2 in direct and nested PCR, respectively (1,3). Amplifications of the DNA from the symptomatic plants yielded a product of 1.8 kb in direct and 1.2 kb in nested PCR assays. No amplification was observed in symptomless plants of the same age and collected from the same fields. Amplicons were purified, cloned in a pTZ57R/T Vector, and sequenced using a Beckman Coulter 8000 CEQ Genetic Analysis System. Four aligned 16S rDNA sequences (1,845 bp) were found to be all identical and belonging to one species. One sequence was deposited in GenBank under the accession number KC139791. A BLAST similarity search revealed that the sequence shared 99% homology with the sequences of the members of 16SrIX group phytoplasmas, 'Brassica rapa' phyllody phytoplasma (HM559246.1) and Iranian Almond witches'-broom phytoplasma (DQ195209.1) available in GenBank. In addition, iPhyClassifier software (4) was employed to create a virtual RFLP profile. The analysis showed that the RFLP profile of the sesame phytoplasma 16S rDNA sequence is identical (a similarity coefficient of 1.00) to the profile of the 16Sr group IX phytoplasma reference sequence (Y16389). A phylogenetic tree was also constructed using the neighbor joining plot option of the Clustal X program. The sequence clustered together with 16SrIX group phytoplasmas. To our knowledge, this is the first report of a natural infection of sesame by a new phytoplasma species from the 16SrIX group in Turkey. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) C. Ikten et al. Phytopathogenic Mollicutes 1:101, 2011. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Control of mushroom sciarid fly Lycoriella ingenua populations with insect growth regulators applied by soil drench.

Mushroom sciarid fly Lycoriella ingenua (Dufour, 1839) comb. nov., is one of the most common fly pests affecting the cultivation of Agaricus bisporus (Lange) Imbach in Turkey. In this study, eight insect growth regulators (IGRs)--diflubenzuron, flufenoxuron, lufenuron, methoprene, novaluron, pyriproxyfen, teflubenzuron, and triflumuron-were tested for their potential to control L. ingenua populations in two successive growing periods. Treatments were targeted at larvae as soil drenches; treatment efficacy was evaluated by assessing adult emergence and larval damage. These products were compared with a control treated with water (negative control) and a conventional chemical insecticide (chlorpyrifos ethyl) (positive control). Treatments with the IGRs caused significant reductions in emerging adult numbers and sporophore damage rates compared with the water-treated control over the two growing periods. Of the IGRs tested, novaluron, diflubenzuron, and teflubenzuron had significantly lower numbers of emerging adults than the rest of the IGRs and chlorpyrifos ethyl-treated control in both periods. Treatments with teflubenzuron, pyriproxyfen, novaluron, and diflubenzuron resulted in significantly lower sporophore damage rates than all other treatments. Compared with negative control, there were no significant yield reductions due to applications of selected IGRs. The results suggest that all the IGRs tested can be used as alternatives to conventional pesticides in controlling L. ingenua populations on mushroom.

Evaluation of resistance to Rhabdocline needlecast in Douglas fir variety Shuswap, with quantitative polymerase chain reaction.

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction.

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.

First Report of Anthracnose Caused by Elsinoë ampelina on Grapes in Michigan.

In 2001, dark brown-to-purple, sunken lesions were observed on shoots and berries of 5-year-old 'Mars' and 'Marquis' table grapes (Vitis spp.) in Onondaga, MI. The disease affected >90% of the vines. Many leaves were curled and distorted and some shoot tips had died back. Older wood showed crater-like indentations. No fruit was harvested due to poor fruit quality. Lesions (at least 10 per sample) were surface-disinfested with 1% sodium hypochlorite, placed on potato dextrose agar (PDA), and kept at 23 to 25°C under ambient light. Sphaceloma ampelinum de Bary (teleomorph Elsinoë ampelina Shear) was isolated from shoots, leaves, and clusters (4). Colonies appeared as slow-growing, dark red mounds. Conidia were hyaline, ovoid, and measured 2.5 to 5 × 5 to 12.5 µm (average 3.5 × 7.4 µm). While grape anthracnose is usually considered a southern disease, occasional outbreaks have been reported in Ohio (1) and it appears to have become more common in Michigan in recent years. Several old herbarium specimens and records of the fungus on grapes in Michigan exist (2,3), but pathogenicity was not proven. In 2004, the disease was confirmed in eight table grape vineyards (three 'Marquis', two 'Concord Seedless', two 'Mars', and one unknown variety), one juice grape ('Niagara'), and one wine grape vineyard ('Vidal') in various locations in Michigan. To determine pathogenicity, shoots of two potted 'Mars' plants were sprayed until runoff with a suspension of 106 conidia/ml of an isolate from 'Marquis' grapes in Lawton, MI. The plants were covered with clear plastic bags for 48 h and kept at 23 to 25°C on the lab bench for 2 weeks. Shoots sprayed with water served as controls. After 4 days, numerous brown spots appeared on inoculated young leaves, petioles, internodes, and tendrils. Lesions continued to enlarge and coalesce into large necrotic areas. The fungus was successfully reisolated on PDA using the procedure described above. ITS1F/ITS4 amplification of three isolates from 'Marquis' vineyards in St. Joseph, Lawton, and Onondaga, MI produced a product of approximately 1,040 bp. The sequences most closely matched that of E. banksiae I. Pascoe & P. Crous from Banksia prionotes Lindl. in GenBank, but no E. ampelina sequences were available for comparison. The sequences of the three isolates in this study were submitted to GenBank (Accession Nos. AY826762, AY826763, and AY826764). The disease in the vineyard in Onondaga may have been present on the plants which originated in Arkansas. Additional evidence for nursery stock as a source of inoculum was the discovery of anthracnose on 'Concord Seedless' plants at a retailer in Okemos, MI in 2004. Anthracnose was also found on riverbank grape (Vitis riparia Michx.) in Holland, MI, indicating the potential of wild hosts to be an inoculum source. However, nothing is known about the ability of isolates from wild grapes to infect cultivated grapes. Control methods, such as the use of disease-free plants, pruning, and effective fungicides are necessary to avoid serious losses in susceptible varieties, particularly in years with heavy rainfall. References: (1) M. A. Ellis and O. Erincik. Anthracnose of grape. Ohio State Univ. Ext. Fact Sheet. HYG-3208-02, 2002. (2) D. F. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory, On-line publication. ARS, USDA, 2005. (3) A. E. Jenkins and A. A. Bitancourt. Nos. 1-550, Myrangiales Selecti Exsiccati, 1940-1963. (4) R. C. Pearson and A. C. Goheen, eds. Compendium of Grape Diseases. The American Phytopathological Society, St. Paul, MN, 1995.