First report of Bursaphelenchus antoniae from Pinus strobus in the U.S.

Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus. Compared to the previously described Portuguese population of B. antoniae, the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus. Compared to the previously described Portuguese population of B. antoniae, the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae. This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.

First Report of Forsythia Shoot Blight Caused by Phytophthora nicotianae in Connecticut.

Blighting of Forsythia × intermedia 'Showoff' was first observed affecting several hundred plants in a commercial nursery in Connecticut in September 2012. Symptoms included wilting, leaf and stem blight, and dieback progressing to plant death. A Phytophthora sp. was isolated from symptomatic tissues on half-strength potato dextrose agar (½PDA). Colonies were white and cottony on ½PDA, reaching 9 mm in 15 days at 25°C, but colorless and inconspicuous on pimaricin, ampicillin, rifampicin, pentachloronitrobenzene agar (PARP) with sparse and limited aerial mycelium, reaching 60 mm in 15 days at 25°C. The characteristics of the pathogen were observed and measured from a 3-month-old colony on ½PDA. Sporangia were abundant, various in shape, ovoid, ellipsoid to pyriform or limoniform, occasionally gourd shaped or irregular; (17.9) 27.2 to 41.4 (47.3) × (12.6) 19.1 to 30.5 (36.7) µm (n = 30), length/breadth ratio 1.4 ± 0.2, papillate and noncaducous. Papillae measured 2.9 ± 0.8 × 3.4 ± 0.8 µm (n = 10). Chlamydospores were present, 23.4 ± 3.1 × 22 ± 3.3 µm (n = 10). Oogonia and oospores were not observed. Arachnoid mycelia were present. These morphological characteristics are consistent with Phytophthora nicotianae Breda de Haan (1). The identity of the pathogen was confirmed as P. nicotianae by BLAST analysis of ITS, Cox II, and beta tubulin gene sequences (99% match for the three sequences, E value = 0). Pathogenicity tests were conducted four times on healthy liners of Forsythia × intermedia 'Showoff' grown in 10-cm-diameter pots. Leaves and stems were wounded by pricking with a sterile needle and six plants were inoculated with 0.25 cm2 plugs of the pathogen growing on ½PDA placed on three leaves and in three stem nodes and covered with Parafilm. Controls consisted of an equal number of plants wounded and inoculated with ½PDA alone. All plants were placed inside high humidity chambers for 24 h and then transferred to a greenhouse for up to 1 month. Typical symptoms developed within 1 week of inoculation and the pathogen was re-isolated from diseased tissue. Disease incidence was nearly 100% of inoculated leaves and stems and not observed in control plants without the pathogen. Three replicate 6-week-old broadleaf tobacco 'C9' plants were each inoculated with tobacco or forsythia isolates of P. nicotianae or sterile media alone, by wounding stems and placing colonized 0.25 cm2 ½PDA plugs into wounds and covering with Parafilm. After 1 week, stems were split and the length of internal necrosis in the stem measured. Disease resulted from inoculation with both the tobacco and forsythia isolates and stem necrosis averaged 43 and 23 mm for tobacco or forsythia isolates, respectively. No necrosis was observed in the pathogen-free controls. P. nicotianae has been reported from the basal stem and roots of F. viridissima in Italy (2) and from shoots of Forsythia × intermedia in Virginia (3). To our knowledge, this is the first report of P. nicotianae causing shoot blight on Forsythia in the northeastern United States. References: (1) J. van. Breda de Haan. Mededeelingenuit's Lands Plantentuin Batavia. 15:57, 1896. (2) S. O. Cacciola et al. Plant Dis. 78:525, 1994. (3) C. X. Hong et al. Plant Dis. 89:430, 2005.

First Report of Downy Mildew Caused by a Peronospora Species on Sweet Basil (Ocimum basilicum) in Massachusetts.

In September of 2008, downy mildew was discovered to be causing a serious foliar blight of sweet basil at several farms and greenhouses in Massachusetts. Infected leaves had chlorotic vein-bounded patches and diffuse chlorosis, and a characteristic gray, fuzzy growth was on the abaxial surface. Microscopic observations revealed branched sporangiophores that measured 187.5 to 325 µm (average 285 µm) long. Sporangia measured 22.5 to 30 × 20 to 22.5 µm (average 26.7 × 20.9 µm). No oospores were found. Sporangium measurements are comparable to unnamed Peronospora species reported previously on basil from Italy, Switzerland, and South Africa (1,2). Sequence analyses were conducted on five isolates of 'Nufar' basil by extracting DNA from a sporangial suspension washed from leaves and infected leaf tissues using the Qiagen DNeasy plant tissue kit (Qiagen, Valencia, CA). PCR amplification of the ITS1, 5.8S, and ITS2 region was performed using primers ITS6 and ITS4 (3). The sequences of the five isolates were identical. BLAST analyses of the sequences revealed a 99% similarity to the unnamed Peronospora species on sweet basil in Europe and South Africa (1,2). To our knowledge, this is the first report of a Peronospora species on sweet basil in Massachusetts. References: (1) L. Belbahri et al. Mycol. Res. 109:1276, 2005. (2) A. McLeod et al. Plant Dis. 90:1115, 2006. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Efficacy of ozone in killing Listeria monocytogenes on alfalfa seeds and sprouts and effects on sensory quality of sprouts.

A study was done to determine the efficacy of aqueous ozone treatment in killing Listeria monocytogenes on inoculated alfalfa seeds and sprouts. Reductions in populations of naturally occurring aerobic microorganisms on sprouts and changes in the sensory quality of sprouts were also determined. The treatment (10 or 20 min) of seeds in water (4 degrees C) containing an initial concentration of 21.8 +/- 0.1 microg/ml of ozone failed to cause a significant (P < or = 0.05) reduction in populations of L. monocytogenes. The continuous sparging of seeds with ozonated water (initial ozone concentration of 21.3 +/- 0.2 microg/ml) for 20 min significantly reduced the population by 1.48 log10 CFU/g. The treatment (2 min) of inoculated alfalfa sprouts with water containing 5.0 +/- 0.5, 9.0 +/- 0.5, or 23.2 +/- 1.6 microg/ml of ozone resulted in significant (P < or = 0.05) reductions of 0.78, 0.81, and 0.91 log10 CFU/g, respectively, compared to populations detected on sprouts treated with water. Treatments (2 min) with up to 23.3 +/- 1.6 microg/ml of ozone did not significantly (P > 0.05) reduce populations of aerobic naturally occurring microorganisms. The continuous sparging of sprouts with ozonated water for 5 to 20 min caused significant reductions in L. monocytogenes and natural microbiota compared to soaking in water (control) but did not enhance the lethality compared to the sprouts not treated with continuous sparging. The treatment of sprouts with ozonated water (20.0 microg/ml) for 5 or 10 min caused a significant deterioration in the sensory quality during subsequent storage at 4 degrees C for 7 to 11 days. Scanning electron microscopy of uninoculated alfalfa seeds and sprouts showed physical damage, fungal and bacterial growth, and biofilm formation that provide evidence of factors contributing to the difficulty of killing microorganisms by treatment with ozone and other sanitizers.

Epipremnum, a new host for Phytophthora capsici.

From 1999 to 2001, a Massachusetts nursery received a number of shipments of Pothos, Epipremnum aureum (Lindl. & André) Bunting, with significant crown, petiole, and leaf rot. The plants were imported from Costa Rica. Sporangia were observed on diseased tissues, and five presumptive isolates of Phytophthora were recovered from infected petioles and stems for species identification. The five isolates were morphologically indistinguishable from each other. Sporangia were produced in water and on V8 juice agar under fluorescent light at 22°C. Mating type was determined by pairing isolates with A1 and A2 mating types of Phytophthora capsici Leonian. Sporangial measurements were taken from water cultures. Determination of caducity, and measurements of pedicels and oospores were taken from V8 agar cultures. Measurements represent an average of 50 observations a single isolate. In water culture, sporangia were borne in umbellate clusters. Sporangium length/breadth was 48.29 and 22.33 µm respectively; length/breadth ratio 2.16. On solid media, sporangia were upright and caducous. The bases of the sporangia were mostly tapered. Pedicel lengths were 22 to 49 µm (average 35 µm). Oogonia had amphigynous antheridia and developed only in the presence of an opposite mating type, and oospores measured 25.74 µm diameter. All five isolates were the A1 mating type. Chlamydospores were absent in V8 and corn meal agar (CMA) cultures. Metalaxyl sensitivity was determined at 0, 0.1, 0.5, and 5 ppm in CMA with five replications. The isolate was completely sensitive to 5 ppm metalaxyl, but grew as well as the controls at 0.1 ppm metalaxyl. Growth response to temperature was determined on V8 agar at 15, 20, 25, 30, and 35°C in five replications. After 4 days, colony diameters at 20, 25, and 30°C were not significantly different (P = 0.01) and colonies filled the 100-mm petri dishes. At 15 and 35°C, average colony diameter was 65.7 and 71.4 mm, respectively. Based on the above characteristics, the isolates were identified as P. capsici. Koch's postulates were carried out on pepper, Capsicum annuum 'Italia', squash, Cucurbita pepo 'Patty Pan' seedlings, and rooted cuttings of pothos. Pepper and squash seedlings and rooted pothos were transplanted in 4-in. (10 cm) pots containing a soilless growing medium (Metro Mix 360, W.R. Grace, Columbia, MD). Phytophthora cultures were grown on V8 juice agar for 4 days. An agar culture was added to 200 ml of sterile distilled water and briefly blended. Ten milliliters of the resulting mycelial slurry were pipetted in the soil one cm from the crown on two sides of the plant. Controls received no mycelial slurry. Petiole, leaf, and crown rot of pothos developed within 2 weeks following inoculation. Squash and pepper plants did not become diseased. In a second pathogenicity test, a 1-cm-diameter plug of mycelial growth from a V8 agar culture was placed between the stem and petiole of the lowest leaf of pothos cuttings directly after transplanting. Inoculated plants died within 3 days. The development of umbellate clusters of sporangia, sporangial shape, length/breadth ratio, and lack of pathogenicity to pepper suggest that the P. capsici isolated from pothos belong to the CAPB (tropical) subgroup of Mchau and Coffey (2). References: (1) S. S. A. Al-Hedaithy and P. H. Tsao. Mycologia 71:392, 1979. (2) G. R. Mchau. and M. D. Coffey. Mycol. Res. 99:89, 1995.

Recent Outbreak of Fusarium Wilt of Basil in Florida.

Symptoms of wilt, leaf chlorosis, leaf drop, and shoot and plant death were observed in commercial fields of basil (Ocimmum basilicum L.). Disease incidence ranged from 10 to 80% among individual fields. Initial isolations from infected stem tissue were made on water agar amended with streptomycin sulfate. Single-spore isolates transferred onto corn leaf agar were identified as Fusarium oxysporum Schlechtend.:Fr. f. sp. basilicum Dzidzariya. Pathogenicity tests were performed on 10-cm-tall basil plants, cv. Siam Queen, for three Florida isolates and one Massachusetts isolate. An inoculum concentration of 1 × 106 conidia per ml was applied to soil around the roots. Symptoms of wilt, external stem discoloration, and death of basil occurred after 14 days, and F. oxysporum f. sp. basilicum was reisolated from plants inoculated with all four isolates. Controls were disease-free. Identification of the isolates as F. oxysporum f. sp. basilicum was done with a set of DNA primers developed by Pan and Wick (2) for a 0.7-kb DNA fragment unique to this pathogen. This report confirms the existence of F. oxysporum f. sp. basilicum in Florida (1), and identifies this disease as a potential threat to commercial basil production. References: (1) S. A. Alfieri et al. Diseases and Disorders of Plants in Florida. Bull. No. 14. Fla. Dept. Agric. Consumer Serv., 1993. (2) Z. Pan and R. L. Wick. Phytopathology 85:1559, 1995.

Evaluation and Identification of Basil Germ Plasm for Resistance to Fusarium oxysporum f. sp. basilicum.

Growth chamber evaluation of several cultivars of basil and related herbs examined in the United States revealed that identical cultivars from different sources did not differ in their reactions to artificial inoculation with Fusarium oxysporum f. sp. basilicum. Cultivars differed in susceptibility to the pathogen: "Spicy globe" miniature was the most susceptible, and lemon basil (Ocimum basilicum var. citriodorum), Origanum majorana, and Thymus vulgaris were rated as not susceptible. Twenty isolates of F. oxysporum, originating from stems of diseased basil plants in Israel, were pathogenic on basil in growth chamber and greenhouse tests. Under artificial inoculation, 2 isolates of F. oxysporum f. sp. basilicum from stems were pathogenic to basil but not to 9 species representing Lamiaceae, Cucurbitaceae, Solanaceae, and Compositae indicating the specificity of the pathogen to basil. These isolates were used for additional resistance tests. Ocimum basilicum var. purpurascens (Exotic) and var. citriodorum were rated as not susceptible to the pathogen under artificial inoculation. Resistant germ plasm was identified in several basil plants of a local variety originally introduced from the United States and reselected at Newe Ya'ar. Seeds were planted in the greenhouse in naturally highly infested soil. Symptomless plants that survived in naturally infested soil were the source for F1 seeds of resistant germ plasm, which was confirmed by artificial inoculations with both isolates of the pathogen. Further selection tests to improve resistance were conducted up to the F4 generation in infested soil in the greenhouse. All individuals of the present genetic line remained symptomless, while all individual plants of the original susceptible cultivar defoliated 3 weeks after planting into infested soil, suggesting that the resistance may be a single, dominant gene. The causal organism was reisolated only from the susceptible plants and not from the symptomless resistant plants through all the experiments.

Detection of Cucurbit Yellow Vine Disease in Squash and Pumpkin in Massachusetts.

Commercial plantings of summer squash in Charlemont, Franklin County, MA, were decimated in 1999 by 100% incidence of a yellowing disease resembling cucurbit yellow vine disease (CYVD) (1). Both plantings were established in the same field during the third week of May, one with transplants and the second by direct-seeding. Each planting consisted of four 30-m rows each of yellow zucchini (Cucurbita pepo cv. Gold Rush), summer squash (C. pepo cv. Seneca Prolific), and zucchini (C. pepo cv. Condor). Crops were produced organically and pyrethrum was used to control a high infestation of squash bugs, Anasa tristis (De Geer) (Heteroptera:Coreidae), a putative vector of CYVD (3). Just prior to fruit set, during the first two weeks of June, plants began showing symptoms of foliar chlorosis, plant stunting, or both. All of the plants in the field eventually wilted and collapsed. Cross-sections of the below-ground stem and primary root revealed a honey-brown phloem discoloration and healthy appearing xylem, symptoms characteristic of CYVD. Plants yielded marketable fruit for only about 1 week. When plant samples were tested by polymerase chain reaction (PCR) with CYVD bacterium specific primers (2), a band of the expected size for the CYVD bacterium, identified as Serratia marcescens based on 16s rDNA and groE sequence analyses (4), was amplified in every case. Since all plant samples collected were symptomatic and PCR positive for S. marcescens, asymptomatic greenhouse plants were run simultaneously as a control. All control plants tested negative. A third planting, similar to the two disease-affected plantings and containing the same three squash cultivars from the same seed lot, was established at about the same time approximately 3 km away. No symptoms of CYVD occurred at this site, further evidence that the pathogen is not seed-borne (1). Furthermore, squash bugs were not observed in this field. In 2000, the disease was observed in a planting of 'Atlantic Giant' pumpkin in Erving, Franklin County, MA, and confirmed by PCR. Until now, CYVD has been reported only in the states of Oklahoma, Texas, and Tennessee. Confirmation of the disease in Massachusetts significantly increases the known geographical range of CYVD to include the New England area. References: (1) B. D. Bruton et al. Plant Dis. 82:512-520, 1998. (2) U. Melcher et al. Phytopathology 89:S95, 1999. (3) S. D. Pair et al. Pages 145-148 in: Proc. 19th Ann. Hort. Conf., Okla. State Univ. (4) J. Rascoe et al. Phytopathology 90:S63, 2000.

The microbiological composition of airliner cabin air.

Hundreds of millions of passengers travel on U.S. airliners annually. These large numbers, together with the close proximity required onboard, raise a concern about microbiologic disease transmission in cabin air. Previous air quality surveys generally concentrated on environmental tobacco smoke and particulate matter. They largely ignored the microorganisms also present. We sampled the microbiologic climate of 45 domestic and international flights. We also sampled common locations in a major southwestern city. The concentration of microorganisms in airline cabin air is much lower than in ordinary city locations. We conclude that the small number of microorganisms found in U.S. airliner cabin environments does not contribute to the risk of disease transmission among passengers.

Hypertension in the civilian flying population: significant or not?

Hypertension is a significant illness that affects a large portion of the population of the United States. We have determined the extent of this illness within the civilian pilot population of the U.S. We reviewed data pertaining to the current prevalence of hypertension in the general population and compared these results to similar data on the pilot population. Though there are some limitations to a direct comparison of these data, we found the pilot population to be significantly different from the general population. The prevalence of hypertension in the general population was 30 times greater than for pilots. Though the overall prevalence in pilots was small, we still consider hypertension to be a significant illness in this group.

A survey of pre-placement urinalysis drug findings.

In December, 1989, the Department of Transportation (DOT) in conjunction with the Federal Aviation Administration (FAA) mandated an extensive urine drug testing program for selected positions within the airline industry. At the end of 1 year we have tested 7,872 applicants under these rules, with a positive finding rate of 0.17%. We have also tested 32,157 applicants, including those applying for DOT-covered positions, with a positive rate of 2.82%. Considering only the two major drugs of abuse--marijuana and cocaine--we found the positive rate to be an order of magnitude greater than the rate discovered under the DOT program. We present these data together with a discussion of some of the possible reasons for this major disparity. We also present findings for barbiturates and benzodiazepines which are not tested under the DOT program, but which have safety implications related to the aviation industry.

Comparisons of pilot performance in simulated and actual flight.

Five highly experienced professional pilots performed instrument landing system approaches under simulated instrument flight conditions in a Cessna 172 airplane and in a Link-Singer GAT-1 simulator while under the influence of orally administered secobarbital (0, 100, and 200 mg). Tracking performance in two axes and airspeed control were evaluated continuously during each approach. The data from the airplane and simulator were compared. Error and RMS variability were about half as large in the simulator as in the airplane. The observed data were more strongly associated with the drug level in the simulator than in the airplane. Further, the drug-related effects were more consistent in the simulator. Improvement in performance suggestive of learning effects were seen in the simulator, but not in actual flight. It is concluded that the GAT-1 simulator is a useful and sensitive device for studies of the effects of mild stress on pilot performance, but extrapolation of simulator data to the flight environment must be approached with considerable caution.

A survey of blood lipid levels of airline pilot applicants.

Cardiovascular disease represents the single largest cause of premature career termination for airline pilots--an entity approximately equal to all other medical causes combined. It is obviously essential to assess risk factors for the development of cardiovascular disease among airline pilots. For that reason, we obtained lipid levels for 14,448 pilot applicants examined during the period from March 1984 through December 1988. Blood lipid levels are well-documented predictors for future cardiovascular diseases. We determined total cholesterol values, high density lipoprotein (HDL) levels, and the cholesterol/HDL ratio for these lipids for our applicant population. We present these data in tabular and graphic form. They suggest a cessation of any increase in blood lipid levels during the fifth decade of life. While the cause is not obvious, there are a number of factors which may play a role in this finding.