First Report of Verticillium Wilt of Ailanthus altissima in Virginia Caused by Verticillium nonalfalfae.

Ailanthus altissima (Mill.) Swingle, commonly known as tree-of-heaven, is an invasive tree species that has spread throughout the United States since its introduction in 1784 (2). During a survey in July 2009, approximately 1,100 A. altissima trees were observed at two locations in western Virginia (a roadside in Montgomery Co. and a wooded area adjacent to a railroad in Bedford Co.) exhibiting foliar wilt symptoms, defoliation, yellowish vascular discoloration, or death at an incidence of ~77%. Similar symptoms on A. altissima were reported in Roanoke, VA in the early 1930s and after 2005 in Pennsylvania, attributed to a Verticillium sp. (1,2). To identify the causal agent, discolored xylem tissue samples were excised from 10 symptomatic A. altissima trees at both locations, soaked in 1% NaOCl for 2 min, rinsed with sterilized distilled water for 5 min, and placed onto plum extract agar. Cultures were incubated in the dark at 22°C for 7 to 14 days. The resultant colonies (three to four per location) were subcultured and identified putatively as a Verticillium sp. closely related to Verticillium albo-atrum Reinke and Berthold (3), based on melanized, thick-walled, resting mycelia and phialides arranged in verticillate whorls that amassed round, oval-shaped conidia (5.1 ± 1.2 µm × 2.8 ± 0.4 µm, n = 100). Molecular identification of two fungal isolates (one per location) was determined by amplification of the protein coding genes elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS), using PCR primers developed recently for Verticillium (3). A BLAST search on the edited contigs revealed 100% sequence similarity for all three protein coding genes among the two isolates and reference sequences of isolates PD592 (GenBank Accessions JN188227, JN188163, and JN188035 for EF, GPD, and TS, respectively) and VnAaPA140 (KC307764, KC307766, and KC307768 for EF, GPD, and TS, respectively) of the newly-proposed species, V. nonalfalfae (formerly V. albo-atrum). Aligned sequences from one representative isolate, VnAaVA2 (Bedford Co.), were deposited into GenBank as KC307758 (EF), KC307759 (GPD), and KC307760 (TS). To confirm pathogenicity to A. altissima, the two molecularly characterized isolates (one per location) were inoculated into 18 10-week old A. altissima stems that were grown in an environmental chamber at 24°C, 60% RH, and a 12-h photoperiod from seeds collected in Blacksburg, VA. A conidial suspension of each isolate was injected into each stem (0.1 ml of 1 × 108 CFU/ml/stem). All 36 seedlings inoculated with the proposed V. nonalfalfae isolates developed wilting of leaflets within 2 weeks post-inoculation (WPI), defoliation of leaflets by 6 WPI, and were dead by 9 WPI. Eighteen control seedlings were inoculated similarly with distilled water, and remained asymptomatic. Fungi resembling the proposed species V. nonalfalfae were reisolated from all inoculated stems except the control plants, and the species confirmed morphologically as described above. V. nonalfalfae is a recently proposed species that can infect a variety of plant species (3). To our knowledge, this is the first report of this proposed species on A. altissima in Virginia. New state reports of this pathogen on A. altissima are important for regulatory issues associated with using this pathogen as a potential biological control agent. References: (1) G. F. Gravatt and R. B. Clapper. Plant Dis. Rep. 16:96, 1932. (2) M. J. Schall and D. D. Davis. Plant Dis. 93:747, 2009. (3) P. Inderbitzin et al. PLoS ONE, 6, e28341, 2011.

Widespread Distribution of Fungivorus Aphelenchoides spp. in Blight Cankers on American Chestnut Trees.

Previously we showed in laboratory studies that the fungivorus nematode, Aphelenchoides hylurgi, was attracted to and fed upon the chestnut blight fungus, Cryphonectria parasitica, from American chestnut bark cankers and was a carrier of biocontrol, white hypovirulent C. parasitica strains. In the present field study, we recovered Aphelenchoides spp. in almost all (97.0 %) of 133 blight canker tissue assays (three 5-g samples each) from four eastern states. High mean population densities (227 to 474 nematodes per 5 g tissue) of Aphelenchoides spp. were recovered from cankers in Virginia, West Virginia, and Tennessee but not from New Hampshire (mean = 75 nematodes per 5 g tissue). Overall, most canker assays yielded population densities less than 200 nematodes per 5 g tissue. All of 12 very small or young cankers yielded a few to many Aphelenchoides spp. Regression analysis indicated greatest recovery of Aphelenchoides spp. occurred in the month of May (r = 0.94). The results indicate that Aphelenchoides spp. appear to be widespread in blight cankers on American chestnut trees and could play a role in biocontrol of chestnut blight.

The effect of fire retardants on combustion and pyrolysis of sugar-cane bagasse.

Experiments were conducted by thermal gravimetric analysis (TGA) and cone calorimetry to measure the affect of three fire retardants (ammonium sulphate, boric acid and borax) on the mass-loss rate and combustion characteristics of sugar-cane bagasse. Compared with untreated bagasse, bagasse impregnated with aqueous solutions of 0.1-0.5M fire retardants exhibited an increase in char mass production from 16% up to 41% when pyrolysed and up to a 41% reduction in total heat release (THR) during combustion. Char mass production was only a weak function of additive concentration over the range of concentrations (0.1-0.5M) used. Combining the additives did not show any synergistic effects for char production or heat release rate (HRR). Treatment of bagasse by these chemicals could be useful to enhance biochar yields in pyrolysis processes or to reduce flammability risk in composites containing bagasse.

Aphelenchoides hylurgi as a carrier of white, hypovirulent Cryphonectria parasitica and its possible role in Hypovirulence spread on blight-controlled american chestnut trees.

Individual nematodes were isolated from American chestnut blight-controlled cankers to determine if they were carriers of biocontrol (hypovirulent) isolates of the chestnut blight fungus, Cryphonectria parasitica. These hypovirulent isolates have a white fungal colony phenotype due to infection by the virus CHV1. Of 1,620 individual Aphelenchoides hylurgi isolated, 29.4% carried propagules of the blight fungus and 8.2% of these yielded white hypovirulent isolates. In attraction and movement tests in Petri plates, A. hylurgi moved 2 cm over 24 hr to mycelial discs of white hypovirulent C. parasitica and pigmented C. parasitica strains in nearly equal numbers. After 2 days of nematode movement to fungal colonies on agar in Petri plates and 21 days of nematode growth, large numbers of A. hylurgi were extracted from both white hypovirulent and pigmented C. parasitica strain colonies. Lower numbers of A. hylurgi were extracted from excised young American chestnut blight cankers that were inoculated with A. hylurgi and incubated for 22 days. A. hylurgi inoculated on the surface of an excised American chestnut canker moved within 24 hr to the small, spore-bearing C. parasitica reproductive structures (stromata) on the canker surface. The results indicate that A. hylurgi may play a role in the spread of hypovirulence on American chestnut trees.

First Report of Mimosa Wilt Disease of Silk Tree (Albizia julibrissin) in California Caused by Fusarium oxysporum f. sp. Perniciosum.

A Fusarium sp. was isolated from a 12-year-old Silk Tree (Albizia julibrissin) in a residential area of Redlands, CA. The scaffold branches and trunk exhibited gummosis, the sap oozing from fissures or intact bark. Internally the wood exhibited brown to black broad streaks of discoloration from the scaffold branches down into lateral roots below the root crown, similar to symptoms observed in Virginia (2). Wilted and dried foliage remained on the scaffold branches. Two-week-old cultures of the isolate grown on Komada (1) and acidified potato dextrose agar media developed short conidiophores, macroconidia, and colony morphology typical of Fusarium oxysporum. To complete Koch's postulates, 1-month-old seedlings were root-dip inoculated with a water suspension of macro- and microconidia (106 per ml). Two weeks after inoculation, typical Fusarium wilt symptoms developed in all inoculated seedlings. The fungus was reisolated from symptomatic seedlings. This is the first report of mimosa wilt disease in California. The disease has the potential to adversely impact California's nursery and landscape industry. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) R. J. Stipes and P. M. Phipps. Phytopathology 65:188, 1975.

The self-retained Le Fort I osteotomy.

Because inferior repositioning of the maxilla after Le Fort I osteotomy has a high relapse rate, a modified bone cut oriented obliquely upward has been tried in 29 patients. The modification allows extrusion of the fragment while maintaining good bony contact, thus eliminating the need for bone grafts. It also permits self-retention of the fragment and can be adapted to movements in any plane.

Low temperature induces decreased germinability of Cylindrocladium microsclerotia.

Numbers of germinable Cylindrocladium crotalariae microsclerotia (assayed at 26 degrees C) decreased progressively over 4 weeks when naturally infested soils were incubated at 6 degrees C. Lower numbers of germinable microsclerotia were obtained from a soil incubated at--3 than at 5 degrees C, while no germinable microsclerotia were found for soils incubated at--10 degrees C. Significantly, when soils incubated at--10 or at 6 degrees C for 4 weeks were transferred to 26 degrees C for 4 weeks, the low-temperature effect was partially reversed. Incubation of naturally infested soils under field conditions from October to February indicated that a similar low-temperature induced phenomenon exists in nature. Germinability of axenic laboratory-grown microsclerotia of C. crotalariae, C. floridanum, and C. scoparium incubated 4 weeks at 6 degrees C ranged from 0 to 91% (mean = 38%) of the initial germinability. Partial recovery of laboratory-grown microsclerotia from the low-temperature effect, by incubation at 26 degrees C, was demonstrated. Conductivity measurements of solutions bathing microsclerotia incubated at 6 degrees C were higher than those incubated at 26 degrees C for 4 weeks and indicated that chilling injury may account, in part, for decreased germinability of microsclerotia.

Improved selective medium for isolating Cylindrocladium crotalariae microsclerotia from naturally infested soils.

Reduced development of fusaria and other undesired fungi, and improved recognition of Cylindrocladium crotalariae colonies on soil-dilution plates were accomplished by supplementing a previously used medium (sucrose-TBZ medium) with three quaternary ammonium compounds, replacing peptone with tyrosine, and increasing the concentration of sucrose to yield a water potential of - 10 bars. These changes eliminated the need to treat soil with NaClO to control undesired fungi. The new medium, designated sucrose-QT medium, allowed increased recovery of C. crotalariae microsclerotia from all 16 naturally infested soils tested.

Roles of low pH, carbon and inorganic nitrogen source use in chlamydospore formation by Fusarium solani.

Citrate and malate were poorer sources of exogenous carbon than several hexose, pentose, or disaccharide sugars for supporting macroconidial germination by Fusarium solani at high conidial density (1 X 10(5) condia/ml). Only citrate, however, failed to block chlamydospore morphogenesis to a degree comparable to glucose or other readily used sugars. Mostly immature chlamydospores were formed in the presence of citrate. At low conidial density (5 X 10(3) conidia/ml), exogenous carbon-independent macroconidial germination and subsequent rapid chalmydospore formation on germ tubes was not inhibited by ammonium or nitrate nitrogen. The citrate-phosphate buffered, low pH (4.0) medium of Cochrane induced more immature chlamydospore formation by F. solani than a pH 6.0 medium, but few mature chlamydospores were formed in either medium. Condensation of hyphal cytoplasm into developing chlamydospores, a character typical of chlamydospore formation, did not occur extensively and macroconidia, hyphae, and immature chlamydospores stained deeply with Sudan III, suggesting lipid biosynthesis. This inhibition of chlamydospore maturation may be due partly to nitrogen deficiency imposed by the high C:N ratio of the medium and to the presence of citrate. Only vesiculate hyphal cells were formed by F. solani f. sp. phaseoli in both media. Field soils to which the clone of F. solani used is indigenous had mean pH values ranging from 5.2 to 6.0.

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.

Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils.

Axenic, washed conidia of Fusarium solani f. sp. phaseoli, Aspergillus flavus, and Verticillium albo-atrum were placed on washed Difco purified agar discs along with an inorganic salt solution containing various levels of carbon and nitrogen substrates. These discs were exposed to volatiles from six soils (pH 5.1-8.6). Fusarium solani macroconidial germination was inhibited mostly by volatiles from soils of pH 5.1, 6.1, 7.0, and 7.5, but high levels of glucose and NH4Cl reversed this inhibition, raising germination to that of no-soil, no-carbon or nitrogen controls. Conidial germination of A. flavus was inhibited mainly by volatiles from high pH (7.0, 7.8, and 8.6) soils, and increased levels of glucose plus an amino acid mixture nullified this inhibition. Volatiles from soils of pH 5.1, 6.1, and 7.5 stimulated A. flavus conidial germination. Assays after the removal of CO2 from the air above soil of pH 5.1 demonstrated that volatiles inhibitory to A. flavus were produced by this soil. Assays indicated that a KOH-soluble compound was a fungistatic soil volatile to F. solani macroconidial germination. The nullification by carbon and nitrogen substrates of F. solani and A. flavus inhibition caused by soil volatiles parallels that for soil fungistasis. Conidial germination of V. albo-atrum was markedly stimulated by volatiles in all soils tested, and was not affected by removal of CO2. Inhibitory soil volatiles may increase the nutritional requirements for spore germination of certain fungi.