Stimulating Duddingtonia flagrans chlamydospore production through dehydration

Duddingtonia flagrans is a nematode-trapping fungus that has shown promising results as a tool to combat parasitic nematode infections in livestock. The fungus interrupts the parasitic lifecycle by trapping and killing larval stages on pasture to prevent re-infection of animals. One barrier to the fungus’ commercial use is scaling up production of the fungus, and specifically of chlamydospores, which survive the digestive tract to grow in fecal pats on pasture, thus have potential as a feed through anthelmintic. The purpose of this study was to evaluate the effect of dehydration on sporulation of the fungus. Disks of Duddingtonia flagrans type strain (ATCC® 13423™) were grown on 17% cornmeal agar for 26 days at 30 °C, then split into three groups; dried quickly at 38 °C and 37% humidity over 48 h (“incubated”), dried more slowly at 24 °C and 55% humidity over 10 days (“air-dried”), or kept at 30 °C and sealed with parafilm to prevent loss of moisture as a control (“wet”). Half of each dried culture was resuspended in water, then heated to liquify and homogenized through vortexing. Spores were then counted in a Neubauer hematocytometer. Both the “air-dried” and “incubated” drying techniques yielded significantly more spores than the “wet” control (Welch’s two sample t test p values of .0359 and .0411, respectively). The difference in average chlamydospores per milliliter was insignificant between the two drying techniques, although a visual representation of the data shows less spore count variability in the “air-dried” technique.

Sugarcane Bagasse: A Potential Medium for Fungal Cultures

Worldwide, sugarcane industries produce tons of sugarcane bagasse as residual/waste material. This residual material is rich in complex lignocellulosic substances and may be used as a low cost carbon and energy source for the growth of fungal species. The present work was aimed at designing a sugarcane waste-based medium as a substitute for expensive commercial media for growing fungal cultures. Eight species of fungi, namely, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Fusarium sp., and four unidentified species F1, F2, F3, and F5, were grown on the sugarcane bagasse medium which produced remarkable results and competed with standard media like potato dextrose agar, Sabouraud dextrose agar, and cornmeal agar. The designed medium was able to provide nourishment to the fungi as well as prevent the growth of any bacterial or fungal contaminant. The production of spores was more in the sugarcane medium as compared with standard media. Hence, this study led to the discovery of a new and efficient medium for fungal cultures as well as decrease in the waste disposal expenses and efforts.

Morphological characterization and identification of phytophthora species causing citrus gummosis in Kenya

Frequent outbreaks of citrus gummosis in Kenyan citrus orchards have been reported, yet the identity and distribution of the Phytophthora species causing the disease are unknown. Work was carried out to (i) characterize and identify Phytophthora species associated with citrus gummosis based on cultural and morphological traits and (ii) determine the distribution of these species associated with gummosis in different agroecological zones (AEZ). Some 59 plant and soil samples obtained from symptomatic trees and the rhizosphere were evaluated by direct isolation and baiting, respectively, using Phytophthora semi -selective media. Phytophthora species were identified on the basis of colony morphology, mycelial characteristics, cardinal growth temperatures, morphology and dimensions of sporangia, oogonia and antheridia. For colony morphology and growth temperature studies, a 5 mm diameter mycelial plug of each isolate was transferred to amended cornmeal agar (ACMA) and incubated at 5, 24 and 35°C for 7 days in the dark. Growth rates were evaluated based on daily records of mycelial growth for 7 days. The occurrence and distribution of these species were determined by recording the number of isolates recovered from samples from each AEZ. P. citrophthora was the most prevalent (76.3 %) of all the Phytophthora species identified in all the AEZs, followed by P. nicotianae (22 %). P. syringae was the least (1.7 %) prevalent. P. citrophthora was the only species present in all AEZs sampled whereas P. nicotianae was confined to the coastal lowlands although also present in other zones in a lower scale. P. syringae was present only in low midland zones and was the only species not found in coastal lowland zones. The forty five isolates of P. citrophthora , thirteen isolates of P. nicotianae and one isolate of P. syringae were tested for virulence on fruits of lemon var. rough lemon. The three most virulent isolates of P. citrophthora , two most virulent isolates of P. nicotianae and the only isolate of P. syringae were selected for pathogenicity testing on lemon seedlings. Based on these studies, it may be concluded that P. citrophthora, P. nicotianae (syn. P. parasitica) and P. syringae are the Phytophthora species associated with citrus gummosis in Kenya. Molecular characterization of the pathogens is recommended to confirm true genetic identity of the species.

Is there ethological isolation among Drosophila melanogaster strains reared for more than 35 generations on different food?

Ethological isolation among three strains of D. melanogaster reared for more than 35 generations on different food (standard cornmeal-agar-yeast medium, banana, and tomato) was tested in a series of multiple choice tests. Long-term maintenance on different substrates did not contribute to sexual isolation among three 'nutritional strains', as degrees of ethological isolation (Malogolowkin-Cohen's isolation indices) were not statistically significant. Males and females reared on different nutritional resources were equally successful in matings, i.e., significant differences in the number of copulations achieved by males and females from various lines were not observed.

Soybean Response to Flooded Soil Conditions and the Association with Soilborne Plant Pathogenic Genera

The role of soilborne pathogens in flood damage on soybeans, Glycine max, was examined using six genotypes representing a reputed range of flood tolerances. Genotypes were planted in single-row plots from 1996 to 1998 with flood treatments of no flood, flood at emergence (3-day duration), or flood at the fourth leaf node growth stage (7-day duration). Three or four days after removing each flood treatment, plant stands were estimated and 15 plants were collected from each plot, weighed, and rated for root discoloration. Roots were assayed for the presence of fungi and other filamentous eukaryotic organisms. Plant stands were reduced by flooding at emergence compared with the nonflooded control. Flooding at both growth stages caused significant increases in root discoloration compared with nonflooded soybeans. Plant weights were reduced in 2 of 3 years for flooding at emergence. Pythium was the only genus of filamentous organisms whose isolation frequency increased with flooding. Of the 60 Pythium isolates evaluated representing the different cultural groups based on appearance and growth rate on potato dextrose agar, cornmeal agar, and V8 agar, 47% were moderately to highly virulent on soybean. Pythium spp. isolated from soybean included the pathogenic species P. ultimum, P. aphanidermatum, P. irregulare, and P. vexans and Group HS. Nonpathogenic P. oligandrum also was isolated from soybean.

Johnsongrass, Yellow Foxtail, and Broadleaf Signalgrass as New Hosts for Six Species of Bipolaris, Curvularia, and Exserohilum Pathogenic to Bermudagrass

Johnsongrass (Sorghum halepense (L.) Pers.), broadleaf signalgrass (Brachiaria platyphylla (L.) Beauv.), and yellow foxtail (Setaria glauca L.) are common volunteer grasses in bermudagrass (Cynodon dactylon (L.) Pers.) pastures in the southeastern United States. Johnsongrass and broadleaf signalgrass are potential forages whereas yellow foxtail is a noxious weed. In 1999 and subsequent years, necrosis and dieback of leaves, stems, and roots, stunting, and plant death were observed on all three species in bermudagrass pastures in north Mississippi (3). Symptoms on johnsongrass and yellow foxtail were most severe where bermudagrass exhibited severe symptoms of infection caused by dematiaceous hyphomycetes (2,3); symptoms on broadleaf signalgrass often occurred independently. Symptomatic leaf tissues from 15 to 33 plants of each species and stem and root tissues from 4 to 14 plants of johnsongrass and yellow foxtail were surface disinfested, plated on water agar, and examined for sporulation after 5 to 10 days (2,3). Pathogens were identified by specific morphological features of spores and sporulation as on bermudagrass (3), and axenic cultures were established by spore transfers to cornmeal agar. Bipolaris cynodontis (Marig.) Shoemaker, Curvularia lunata (Wakk.) Boedijn, C. geniculata (Tracy & Earle) Boedijn, and Exserohilum rostratum (Drechs.) Leonard & Suggs were isolated from symptomatic leaves of all three grasses and frequently also observed on stems and roots. B. stenospila (Drechs.) Shoemaker was observed only on broadleaf signalgrass (19 of 33 plants) and B. spicifera (Banier) Subr. on johnsongrass and yellow foxtail. Species most frequent on leaves (58 to 100%) were B. spicifera, C. lunata, and E. rostratum on johnsongrass and yellow foxtail and B. cynodontis, B. stenospila, and E. rostratum on broadleaf signalgrass. The three grasses were grown from seed in potting mix in the greenhouse (one plant per 375-cm3 container), and five replicates 31 to 60 days old were inoculated with a mixture of three isolates of each pathogen observed on them in two experiments. Conidia produced from infested wheat and oat grain were atomized onto foliage (1.2 to 4 × 104 conidia per ml, 20 ml per plant) as described (2). All pathogens incited similar necrotic lesions and streaks on the three grasses after 12 to 15 days, and B. stenospila also caused extensive golden yellow chlorosis on broadleaf signalgrass. All pathogens caused significant (P = 0.05) necrosis (means = 5 to 35% of foliage necrotic based on visual estimates, controls = 1 to 3%), and all were reisolated and grown in pure culture by spore transfers to cornmeal agar from surface-disinfested, symptomatic leaf tissue of each grass. When bermudagrass grown from seed was inoculated at similar spore concentrations, isolates of E. rostratum, B. cynodontis, and B. spicifera from two or all three grasses caused symptoms as severe as did isolates from bermudagrass. Results document new North American or worldwide records of occurrence and pathogenicity for B. cynodontis, C. geniculata, and C. lunata on all three grasses, B. stenospila and E. rostratum on broadleaf signalgrass, and B. spicifera on johnsongrass and yellow foxtail (1). These volunteer grasses, bermudagrass, and the six fungi all appear to represent large, interacting complexes of multiple hosts and potentially cross-infecting pathogens. Reference: (1) D. Farr et al. Fungal Databases. Systematic Botany and Mycology Laboratory. Online publication. USDA, ARS, 2005. (2) R. Pratt, Agron. J. 92:512, 2000. (3) R. Pratt. Phytopathology 95:1183, 2005.

(97) Screening Pythium sp. Associated with Greenhouse Crops in Colorado for Mefanoxam Resistance

Research was conducted on the fungus-like organism Pythium to observe its sensitivity to the fungicide Subdue MAXX, active ingredient mefanoxam. Pythium is a plant pathogen that causes root and crown rot in plants that are exposed to extensive soil moisture and poor drainage. This study, conducted on Pythium isolated from Colorado greenhouse crops, began in Apr. 2004. Symptomatic tissues from bedding plants and perennials were placed on selective media (P10VP). Resulting isolations of Pythium were transferred to cornmeal agar amended with the recommended low and high label rates of mefanoxam. Mycelia growth was monitored for 72 hours. Pythium sp. showing 20% growth on amended mefanoxam agars, compared to the control growth, were considered resistant to mefanoxam. Results from this study showed that about 64% of the Pythium sp. isolated from greenhouse crops in Colorado are resistant to mefanoxam. Data compiled from greenhouse integrated pest management surveys in Colorado show a high dependence on the use of mefanoxam. Research and screening for mefanoxam-resistant Pythium sp. will continue to provide Colorado growers with effective control solutions for this pathogen.

FUNGICIDAL PROPERTIES OF GARLIC EXTRACT IN Sphagnum PEAT- AND SAND-BASED SUBSTRATES

Growth of Pythium aphanidermatum, Pythium ultimum, Pythium irregulare, Phytophthora nicoctianae, Phytophthora cinnomomi, Fusarium oxysporum, Rhizoctonia solani and Thielaviopsis basicoli was inhibited in vitro when grown in a clarified V-8 nutrient solution containing 10% garlic extract. After exposure to 10% garlic extract for 3 days, all fungi and fungal-like organisms failed to grow after being washed and transferred to fresh cornmeal agar nutrient medium without garlic extract. When Sphagnum peat was inoculated with P. aphanidermatum and drenched with solutions containing varying concentrations of garlic extract, a single drench of 35% garlic extract or two drenches of 15% garlic extract were required to rid the substrate of viable P. aphanidermatum. In sand, a single application of 25% garlic extract or two applications of 10% garlic extract were required to rid the sand of viable P. aphanidermatum Thus, Sphagnum peat appeared to partially inactivate the components in garlic and did so to a greater extent than sand. Therefore, efficacy of garlic extract as a soil drench fungicide will be affected by the type of substrate or soil to which the garlic extract is applied.

Pythium Species Associated with Root Dysfunction of Creeping Bentgrass in Maryland

Putting green samples (n = 109) were inspected for the presence of Pythium oospores in roots of plants from golf courses (n = 39) in Maryland and adjacent states. Twenty-eight Pythium isolates were recovered from creeping bentgrass (Agrostis palustris) (n = 25) and annual bluegrass (Poa annua) (n = 3) plants. Most isolates associated with Pythium-induced root dysfunction were from greens less than 3 years of age and were obtained primarily between March and June, 1995 to 1997. Eight Pythium species (P. aristosporum, P. aphanidermatum, P. catenulatum, P. graminicola, P. torulosum, P. vanterpoolii, P. volutum, and P. ultimum var. ultimum) were isolated from creeping bentgrass and two species (P. graminicola and P. torulosum) were from annual bluegrass. All species, except P. catenulatum, were pathogenic to ‘Crenshaw’ creeping bentgrass seedlings in postemergence pathogenicity tests. P. aristosporum (n = 3) and P. aphanidermatum (n = 1) were highly aggressive at a low (18°C) and a high temperature (28°C). P. graminicola (n = 1) was low to moderately aggressive. P. torulosum (n = 12) was the most frequently isolated species, but most isolates were either nonpathogenic or caused very little disease. P. aristosporum (n = 3) and P. aphanidermatum (n = 1) were highly aggressive and were associated with rapid growth at 18 and 28°C on cornmeal agar. P. volutum (n = 1) was highly aggressive at 18°C, but was one of slowest growing isolates. Infected roots were generally symptomless, and the number of oospores observed in roots was not always a good indicator of disease or of the aggressiveness of an isolate. Large numbers of oospores of low or even nonpathogenic species may cause dysfunction of creeping bentgrass roots.

Isolation, Characterization, and Distribution of a Biocontrol Fungus from Cysts of Heterodera glycines

Seventy-six populations of Heterodera glycines were collected from 33 counties in 10 states of the United States along the Mississippi and Missouri Rivers in 1992 and 1993. A sterile hyphomycete fungus of an unnamed taxon, designated ARF18 and shown to be a parasite of eggs of H. glycines, was isolated from eggs and cysts of 10 of the populations from Kentucky, Louisiana, Mississippi, and Tennessee. Ten isolates of ARF18 obtained in this study and seven isolates obtained in earlier studies were characterized for cultural morphology on several growth media, the ability to produce sclerotium-like structures (SLS) on cornmeal agar, growth rates, pathogenicity to eggs of H. glycines in vitro, and mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs). All 17 isolates of ARF18 readily grew on potato dextrose agar, cornmeal agar, and nutrient agar. Based on colony morphology and SLS appearance on cornmeal agar, the isolates could be grouped into two morphological phenotypes. Isolates that produced SLS that were composed of a compact mass of hyphae were designated ARF18-C, whereas isolates that produced SLS composed of a mass of loosely clumped hyphae were designated ARF18-L. Only minor differences in growth rates were detected among the ARF18-C and ARF18-L isolates. All 10 ARF18-C isolates, which were from Arkansas, Louisiana, Mississippi, and Tennessee, belonged to a single mtDNA RFLP haplotype. The seven ARF18-L isolates shared many comigrating mtDNA restriction fragments with one another, but belonged to three distinct mtDNA RFLP haplotypes. Ability to infect eggs of H. glycines in vitro varied considerably among the various isolates of ARF18. In particular, several of the ARF18-C isolates were consistently able to infect over 50% (mean = 70.0%, standard deviation = 16%) of the eggs of H. glycines, whereas ARF18-L infected eggs to a lesser degree (mean = 25%, standard deviation = 27%). ARF18-C was isolated only from H. glycines populations from below 37° N latitude. The presence of ARF18 was associated with soils with Mg levels <314 kg/ha, cyst numbers >4.5 per 100 cm3, and iron levels >203.5 kg/ha; or with Mg levels >314 kg/ha and Na levels <121 kg/ha. The widespread distribution of ARF18 and the ability of some isolates to aggressively colonize eggs of H. glycines are indications that it has potential as a biological control agent for H. glycines.