Water Agar Plates Research Articles

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.

Antibiosis, mycoparasitism, and colonization success for endophytic Trichoderma isolates with biological control potential in Theobroma cacao

Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-induced diseases. Cacao supports a complex endophytic microbial community that offers candidates for biocontrol of cacao diseases. Endophytic isolates of Trichoderma species were isolated from the live sapwood of trunks of Theobroma species, pods of Theobroma species, and a liana ( Banisteriopsis caapi). Fifteen isolates of Trichoderma, potentially representing seven species, were selected for characterization of the influence of seedling inoculation on the establishment of endophytic growth in cacao seedlings. An isolate of Colletotrichum gloeosporioides was also included. The isolates studied in vitro varied in their abilities to produce metabolites inhibitory to Moniliophthora roreri and in their abilities to parasitize M. roreri cultures. The five inoculation methods used were: (1) inoculation of germinating seed on agar plates; (2) plate inoculation followed by planting in sterile soil; (3) planting sterile seed in pre-inoculated soil; (4) inoculation of emerged seedlings at the soil surface; and (5) inoculation of emerged seedlings between the cotyledon and stem. All the isolates studied were able to colonize Theobroma cacao seedlings, but isolates DIS 110a ( Trichoderma cf. harzianum), DIS 219b ( T. hamatum), DIS 219f ( T. harzianum), and TA ( T. asperellum) were the most efficient across inoculation methods. These same isolates also caused moderate to severe discoloration of roots of cacao seedlings germinated on water agar plates. Isolates DIS 173a ( T. spirale), DIS 185c ( T. stromaticum), and Col ( Colletotrichum gloeosporioides) were inefficient colonizers of cacao. Most of the isolates studied were able to establish an endophytic relationship with cacao by colonizing the above ground portions of the cacao seedling, and exploitation of this characteristic could lead to the development of novel biocontrol strategies for control of cacao diseases.

Tilletia indica

<i>Tilletia indica</i>

Diaporthe eres (Phomopsis oblonga) as a Pathogen of Butternut (Juglans cinerea) in Connecticut

Butternut seedlings from North Carolina (S. Schlarbaum, University of Tennessee) were planted during the spring of 2005 in an open space at the edge of the Mattatuck State Forest in Plymouth, CT. There were mature butternuts with visible cankers that were at the forest edge and out of reach. In May 2006, some seedlings had died and many small, dark lesions were found on the twigs and stems of living seedlings. Stems with lesions were surface sterilized with 10% bleach, rinsed with sterile water, the epidermis peeled away, and slices of the wood were placed on the surface of 2% water agar plates. From these, we consistently isolated a Phomopsis sp. that grew well on potato dextrose agar (PDA) at 28°C and produced alpha and beta spores (average 8 × 2 μm and 25 × 1 μm, respectively). A mixture of six such isolates on autoclaved pieces of butternut stem supported by water agar produced perithecia after 6 months with ascospores that matched the description of Diaporthe eres (two-celled, slightly constricted at the septum, and averaged 12 × 3 μm). Two of our isolates of Diaporthe eres, both of which were weakly pathogenic and recovered from the lesions, were inoculated into cut, dormant stems of butternut 'Buckley' in the laboratory, This ascomycete was reported to be a pathogen of Juglans ailantifolia and J. regia var. orientes in Japan (1) but has not been reported on J. cinerea. There is a rather vague report of Diaporthe spiculosa on other species of Juglans and Phomopsis juglandina on twigs (with a question mark) of J. regia (with a question mark) in California (2). These were not reported by T. Kobayashi.

Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus Suillus bovinus

Under laboratory conditions, spores of ectomycorrhizal fungi usually germinate very poorly or not at all. In a previous study, we showed that spores of the ectomycorrhizal fungus Suillus bovinus germinated through the combination of activated charcoal treatment of media and co-culture with seedlings of Pinus densiflora, which suggested that some substances contained in root exudates induced the germination. Among the compounds reported from root exudates, flavonoids have been elucidated to play various and substantial roles in plant-microbe interactions; we therefore investigated the effects of flavonoids on basidiospore germination of S. bovinus by the diffusion gradient assay on water agar plates pretreated with charcoal powder. Seven out of the 11 flavonoids tested, hesperidin, morin, rutin, quercitrin, naringenin, genistein, and chrysin, had greater effects than controls, whereas flavone, biochanin A, luteolin, and quercetin showed no positive effects. The effective concentration presumably corresponded to several micromolar levels, which was equivalent to those effective for pollen development, nod gene induction, and spore germination of F. solani f. sp. pisi and AM fungi. The results suggest that flavonoids play a role as signaling molecules in symbiotic relationships between woody plants and ectomycorrhizal fungi.

Attraction of the pinewood nematode to mycelium of some wood-decay fungi

Migration of the pinewood nematode, Bursaphelenchus xylophilus, to the mycelia of wood-decay fungi was investigated on water agar plates. A very strong ability to attract nematodes was shown by Pleurotus ostreatus, P. pulmonarius, P. eryngi, Lentinula edodes and Lampteromyces japonicus, all of which had been demonstrated to be nematophagous in previous studies. Other wood-decay fungi, Neolentinus lepideus, Trichaptum abietinum, Cryptoporus volvatus and Pholiota nameko, having less or no ability of killing nematodes, did not attract nematodes. Botrytis cinerea attracted nematodes although not as strongly as the nemtophagous wood-decay fungi. Based on the experimental results it could be concluded that nematophagous wood-decay fungi were attractive to the pinewood nematode.

First Report of Ramularia cercosporelloides on Carthamus tinctorius in Northwestern Mexico.

Severe yield losses caused by an unidentified fungal disease occurred on safflower in Sonora, Mexico from 2001 to 2006. Leaf spots were pale at the beginning and became sunken lesions, spots turned brown, and diseased tissue became necrotic. Under continuous presence of dew, new infections occurred and the number of lesions increased, coalesced, and eventually the entire leaf and plant turned brown and dried up. The fungus appears as a whitish mold covering the lesions. Observations with a dissecting microscope revealed fungal growth on both leaf surfaces. Spots had minute, gray-to-pink tufts emerging from the host tissues. Stromata were roughly spherical, 35 to 55 μm in diameter, developed under the epidermis near the edge of the spots from which conidiophores arose. Fascicles of conidiophores emerged through stomata from colorless or slightly yellowish stroma and were simple, colorless, or slightly yellowish, as much as 85 μm long and 2.5 to 3 μm wide, with a pigmented scar at the tip. Conidia, formed in chains at scar sites on conidiophores, were colorless, obclavate, 0 to 1 septate, approximately 24 to 32 μm long, and 4.5 to 7 μm wide at the broadest part with a pigmented scar either at the base or at both ends. On the basis of symptoms and fungal morphology, the pathogen was identified as Ramularia cercosporelloides U. Braun & Crous (=Cercosporella carthami) (1). Seven specimens were deposited at the Uppsala University Herbarium as Nos. UPS F-119998 to 120004. Conidia were transferred to water agar plates from symptomatic leaves after 8 days in a humid chamber. Ten single conidia were plated onto a Septoria tritici medium (4 g each of malt extract, yeast extract, and sucrose, and 18 g of agar per liter of water). Colonies of the fungus were white-to-light pink with irregular margins and very slow in growth. Inoculum was produced by transferring 2 ml of distilled water containing conidia onto petri plates filled with Septoria medium. Plates were incubated at 18°C for 12 days. Water suspension was amended with two drops of Tween 20 per liter of inoculum. Pathogenicity of five isolates was confirmed by spraying a suspension of 1.0 × 105 conidia/ml onto five pots of four direct-seeded adult plants of cv. S-518 per isolate. Plants were kept in a dew chamber (20°C) for 48 h under 16 h of darkness and 8 h of light and then in a greenhouse (20 to 24°C). After 2 weeks, leaves developed the characteristic spots, and R. cercosporelloides was isolated from symptomatic tissue. To our knowledge, this is the first report of R. cercosporelloides on safflower, not only in Mexico, but also on the American continent. Reference: (1) U. Braun. A monograph of Cercosporella, Ramularia and allied genera (Phytopathogenic Hyphomycetes). Vol 2. IHW-Verlag. Eching bei Munchen, 1998.

Teleomorph Formation of Phaeoacremonium aleophilum, Cause of Esca and Grapevine Decline in California.

Phaeoacremonium is a recently described genus of the hyphomycetes and includes species associated with grapevine (Vitis vinifera) declines worldwide. Spores of Phaeoacremonium spp. have been trapped in infested vineyards, but neither asexual nor sexual fruiting structures have been observed in the field. Mating studies were carried out to determine if California P. aleophilum isolates are capable of forming a teleomorph in vitro. Sterilized grapevine shavings were placed on the surface of water agar plates with pairs of different California isolates of P. aleophilum, an isolate from the holotype of P. aleophilum, plus other related Phaeoacremonium spp. After approximately 28 to 35 days, perithecia were seen forming on wood chips and agar of many pairings. Upon maturation, fertile perithecia had gelatinous droplets of ascospores oozing from their ostioles. Successful crosses, resulting in mature perithecia, corresponded to a heterothallic mating type system. When F1 progeny were backcrossed with their parents, heterothallism was confirmed. Molecular analyses of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA from ascospore progeny and perithecia confirmed that these perithecia were the teleomorph of P. aleophilum, Togninia minima. Furthermore, 4 months after moist incubating grapevine pieces from naturally infected vineyards, mature perithecia of T. minima could be seen forming on the xylem and pith tissues, suggesting both mating types occur on the same vine.

Optimisation of a Selection Scheme using Kanamycin to Improve Transformation of Medicago truncatula cv. Jemalong

We developed an efficient method for in vitro selection of Medicago truncatula cv. Jemalong lines transformed with the nptII gene and subsequent confirmation of phenotype inheritance in these lines. For in vitro selection, the concentration of kanamycin inhibitory to embryogenic callus development and somatic embryo differentiation was identified by placing wounded leaves of non-transformed M. truncatula cv. Jemalong on Embryo Inducing Medium supplemented with 0, 85.8, 128.7, 171.6, 214.6, 257.5 and 343.3 µM of kanamycin. Differentiation of somatic embryos was inhibited with 171.6 µM of kanamycin but callus development was not altered. To confirm transgene inheritance, the kanamycin concentration to distinguish between resistant and non-resistant seedlings was identified by germinating non-transformed seeds of M. truncatula cv. Jemalong on 0.8% (w/v) water-agar plates containing 0, 171.6, 343.3, 514.9 and 686.6 µM of kanamycin. These concentrations did not impair seed germination since all the seedlings exhibited green cotyledons. The effect of kanamycin was only observed at 514.9 and 686.6 µM and on the first pair of leaves, which became white. Due to the high level of resistance to kanamycin of the seedlings the highest concentration is thought to be use to assure the selection efficiency. This optimised antibiotic selection scheme eliminates the regeneration of non-transformed escapes and discriminates between resistant and non-resistant seedlings, confirming the inheritance of the phenotype in transformed M. truncatula cv. Jemalong lines.

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

AbstractThe effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m2/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10−4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (−15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.

Description of spore stages of Puccinia sorghi in South Africa

The life-cycle of Puccinia sorghi, a heteroecious fungus, consists of five well-defined spore stages. The uredinial and telial stages are completed on the primary maize host whereas pycnial and aecial stages occur on Oxalis comiculata (creeping sorrel), a perennial and widespread weed. Although the sexual phase of maize rust has been known to occur in South Africa for many years, accurate descriptions of spore stages are not readily available. Surface-sterilised discs of maize leaves containing telia were placed on water agar plates and inverted over O. comiculata plants covered with a settling tower. Removal of plants at different time intervals showed that 6 h of high humidity at 19–21°C were sufficient for germination of teliospores and infection of the alternate host by basidiospores. Both light and scanning electron microscopy of O corniculata leaf sections provided detailed descriptions of the pycnia, containing receptive hyphae and spermatia. and aecia with aeciospores. The frequent detection of aecial infections on O. comiculata in KwaZolu-Natal suggested that sexual recombination may play a rote in generating new variants of P. sorghi in South Africa.

An Excised-Leaf Inoculation Technique for Evaluating Host-Pathogen Interactions and Quantitative Resistance of Bermudagrass Genotypes to Dematiaceous Hyphomycetes

ABSTRACT Excised leaves of bermudagrass were inoculated with mycelium of isolates of Bipolaris, Exserohilum, Curvularia, and Drechslera spp. in water agar plates to evaluate differences in susceptibility of leaf tissue, virulence of pathogens, and quantitative resistance of bermudagrass genotypes. Isolates of nine species of pathogens induced similar symptoms of light- to dark-brown necrosis and bordering chlorosis in excised leaves that were not distinct for individual species or genera. Severity of symptoms induced by most isolates increased progressively from younger to older leaves. Within and across leaf positions, numerous significant differences in virulence of isolates within fungal species and between species were observed. Among 40 randomly selected bermudagrass genotypes, a continuous quantitative gradient was observed for mean scores of disease severity in excised leaves inoculated with E. rostratum. Numerous significant differences were observed within this gradient, and severity of symptoms in the most susceptible genotypes was approximately double that in the most resistant. When intact foliage of genotypes from the resistant and susceptible extremes of the gradient was inoculated with spores of E. rostratum, corresponding differences in severity of symptoms and significant (P = 0.05) correlations between results with excised leaves and intact foliage were observed. However, the range of differences in disease severity between genotypes was more narrow in intact foliage than in excised leaves. Results indicate that the excised leaf inoculation technique can be used to evaluate the relative resistance of bermudagrass genotypes to E. rostratum for use in programs to breed for quantitative host resistance.

Biological control of root rot of cauliflower (caused by Pythium ultimumvar. ultimum) using selected antagonistic rhizospheric strains of Bacillus subtilis

Cauliflower (Brassica oleracea var. botrytis) with a root rot disease were found in a field in Kidwan village, 3 km south of El‐Minia city, Egypt, during January 2001. Cauliflower plants showed wilting soon after transplanting. In addition, cauliflower seedlings collected from the same field showed browning lesions at the basal part of the stem and the root system and eventually led to wilting. Examination of root pieces and other infected parts yielded only one type of fungus characteried as Pythium ultimum var. ultimum by its typically saclike monoclinous and diclinous antheridia and aplerotic oospores. P. ultimum var. ultimum was shown to be pathogenic on cauliflower under glasshouse conditions. This is the first report of P. ultimum var. ultimum causing disease to cauliflower in Egypt. Bacteria producing antibiotics against the root‐rot fungus Pythium ultimum var. ultimum were selected. Agar discs from rhizosphere soil of cauliflower containing colonies were transferred onto water agar plate culture of P. ultimum var. ultimum. After 2 days of incubation, colonies producing clear inhibition zones were readily detected. The two strongest antagonistic bacteria were identified as Bacillus subtilis. Subsequent plant growth experiments established that substantial disease control could be obtained by applying B. subtilis to the soil. Optimal control was obtained by mixing the bacteria with the soil rather than by dipping the cauliflower roots in the bacterial suspension immediately before planting. Disease was more severe in autoclaved soil than in non‐sterile soil.

Direct invasion of bones by highly pathogenic fungi in an in vitro model and its ecological significance.

Animal bones after being devitalized at death are strongly resistant to wear and tear and remain in the soil or environment much longer than other organic components from dead animals. Yet over the course of time they seem to disappear and thus our ecological surroundings are not cluttered with bone remnants. Mechanical factors creating compression or friction and chemical factors like pH of the soil and surroundings must together have provided concerted degrading effects. Microorganisms in the soil also help in this process by utilizing the organic components of devitalized bones. Certain highly pathogenic fungi that have been collected from soil from time to time and many other environmental fungi may take part in the degrading of the bone remnants. In this study, several strains from the highly pathogenic dimorphic fungi Coccidioides immitis, Blastomyces dermatitidis, Histoplasma spp., Paracoccidioides brasiliensis and also some strains of dematiaceous fungi (Exophiala spp. and Foncecaea pedrosoi) were inoculated to dissected and devitalized murine long bones that had been placed on solidified water agar plates to see if they would survive, grow and invade the bones. After being kept for 12 weeks at 25 degrees C all the parts of the histological sections of these bones showed invasion by most of the strains used in this study, although the cortical component of the bony architecture seemed to be comparatively resistant to invasion. Their ability to grow and sporulate in the aforementioned nutrient-limiting condition hinted at a possible role of these fungi in the degradation of devitalized bones.

First Report of Downy Mildew of Quinoa Caused by Peronospora farinosa f. sp. chenopodii in Denmark.

Downy mildew, caused by Peronospora farinosa (Fr.) Fr. f. sp. chenopodii Byford, is the most important disease of quinoa (Chenopodium quinoa Willd.) in the high mountainous areas of the Andes in South America (1), where the crop originated. While quinoa is not well known outside its traditional growing area, interest in this crop is increasing rapidly due to its high nutrient quality, many uses for human consumption, and agronomic characteristics of the plant; it is one of the most drought, salt, and frost tolerant crops known (2). From 1990 to 2001, several field trials were conducted in Denmark to study the performance of quinoa germ plasm of different origin under Danish conditions. Natural infection by downy mildew was observed each year on a range of cultivars, as well as on the common weed species, C. album L., which was observed growing near the quinoa. In 2001, 25 isolates of P. farinosa were collected from a field trial at the experimental station of the Royal Veterinary and Agricultural University in Taastrup, Denmark, from two Dutch quinoa cultivars (Carmen and Atlas) and one Danish breeding line (G205). Plants showed typical downy mildew symptoms: chlorotic lesions on the upper leaf surface and grayish spore masses on the lower leaf surface. Microscopic examination of the material showed the presence of dichotomously branched sporangiophores (200 to 400 μm long), which tapered to a blunt point and produced ellipsoidal, light brown sporangia with mean dimensions of 22 × 18 μm. The isolates were propagated and maintained by inoculation of detached quinoa leaves of two highly susceptible Peruvian cultivars (Toledo and Pandela) with a sporangium suspension (105 sporangia per ml), followed by incubation on water agar plates (10 to 15 leaves per plate) at 15°C and 12 h light/dark for 9 to 11 days. All isolates grew readily on these cultivars, and microscopic examination of the pathogen showed the same morphology as the original isolate. When the isolates were tested for their virulence on a range of quinoa cultivars with different geographic origin, specific interactions between host genotype and pathogen isolate were observed, measured as the degree of sporulation in the detached leaf assay, suggesting the presence of several pathotypes. To our knowledge, this is the first report of P. farinosa on quinoa in Denmark. Downy mildew should be considered as a potential problem for future large-scale quinoa production in Europe.

A TRICHODERMA KONINGII SEED TREATMENT IS EFFECTIVE BEFORE AND AFTER SEED STORAGE AGAINST SCLEROTIUM ROLFSII DAMPING OFF OF TOMATO

An isolate of Trichoderma koningii was used for treatment of tomato seeds with a suspension of 106 con/ml in dilute Tween-20 or sterile distilled water. Seeds were stored at 6°C or 20°C and periodically sampled in order to determine the efficacy of Trichoderma koningii as a biocontrol agent against Sclerotium rolfsii damping off in the greenhouse. Seed germination tested in water agar plate showed that T. koningii slightly delayed seed germination over control treatments. Length of storage had a significant effect on the survival of Trichoderma on seeds, which was more pronounced when seeds were stored at 20°C rather than at 6°C. The biocontrol ability of T. koningii, expressed as pre- and post-emergence damping off in artificially infested sterile potting compost, was affected by the length of storage. Storage temperature also excerted a significant (P=0.034) effect on final stand. After each storage period, Trichoderma-treated seed still gave significantly greater seedling counts of plants than uncoated control seed.

Histopathological examination of in vitro bone degeneration caused by a black yeast, Exophiala spinifera.

Exophiala spinifera, a black yeast, rarely causes systemic infection, and only a very few cases of its infection by the invasion of internal organs or bones have been reported. We examined the ability of E. spinifera to invade bone tissues in vitro. The fungus was inoculated on the surface of murine bones, and then these bones were incubated at 30 C for 2, 4, and 12 weeks on water agar plates and on brain heart infusion agar supplemented with 1%glucose (BHIA) plates. Histopathological examination demonstrated that the fungus was initially found in the non-calcified parts of the bone tissue, such as the growth plate and articular cartilage. Thereafter, the fungus invaded the calcified parts: cancellous and cortical bones. Our experiments showed that the capability of E. spinifera to invade bone tissue is higher than that of Candida albicans or other black fungi. E. spinifera grew in the mycelial form and C. albicans in the yeast form in these experiments. Our results suggest that E. spinifera may have a high potential to invade bone tissues, and that the mycelial form can invade bone more deeply than the yeast form. Therefore, bone degeneration should/ must be carefully monitored in any systemic infection with E. spinifera.

Karnal Bunt of Wheat Newly Reported from the African Continent.

In December 2000 seed harvested from wheat (Triticum aestivum L.) cultivars SST 876 and SST 825 produced under sprinkler irrigation near Douglas, Northern Cape Province, South Africa, contained a substantial amount of partially bunted kernels. Kernel embryos contained black masses of teliospores, and in many instances the endosperm was partially degraded. Teliospores were brown to dark brown, densely echinulate, 25 to 45 μm in diameter with a short mycelial fragment on some of the spores. Hyaline, smooth-walled sterile cells were also present. Teliospores were soaked in sterile distilled water for 2 days, streaked on 2% water agar plates and incubated at 22°C in the dark. Teliospores germinated after 5 days, producing 50 to 250 filiform, nonconjugating, primary basidiospores and forcibly discharged allantoid, secondary basidiospores. Based on kernel appearance, a rotten fish odor in infected grain, teliospore morphology, and germination characteristics, the pathogen was identified as Tilletia indica Mitra, the cause of Karnal bunt (1). This morphological identification was confirmed at the USDA-ARS Systematic Botany and Mycology Laboratory, Beltsville, MD. Molecular verification of 12 South African isolates was provided by the Foreign Disease-Weed Science Research Unit at Fort Detrick, MD, using real-time polymerase chain reaction with the Tin3/Tin10 T. indica-specific primer set (2). Four additional isolates were confirmed as T. indica using the same primer set as well as ITS rDNA sequencing at the Beltsville laboratory. Reference specimens were deposited at the National Fungal Collection in Pretoria, South Africa (PREM 57214), and at Beltsville (BPI 748170). At present, the mode of introduction of T. indica into South Africa, as well as its precise distribution, is not known. It appears, however, that the pathogen is restricted to the Douglas production area in the Northern Cape where quarantine measures have been taken to contain and possibly eradicate the disease.

Occurrence of Puccinia xanthii on Sunflower in South Africa.

A severe outbreak of a foliar disease occurred on ornamental sunflower (Helianthus annuus L. cv. Tyao) cultivated under plastic protection near Potchefstroom in the North West Province during February 2000. According to previous descriptions of host range, symptoms, and spore morphology, the pathogen was identified as the microcyclic rust fungus, Puccinia xanthii Schwein. (2-4). Telia, reaching 9 mm in diameter and often coalescing to form larger lesions associated with chlorosis, occurred on the lower surface of leaves. Symptoms were first observed at the V-12 growth stage when approximately 40% of leaves displayed lesions. The disease subsequently spread to younger plants (V-2 stage) where an incidence of 100% was recorded. Severity estimates regularly reached 50% leaf area affected. The primary source of inoculum was thought to be nearby rust-infected Xanthium strumarium sensu lato (burweed or cocklebur) plants, a known host for P. xanthii (2,4). Infection studies were carried out on 3-week-old sunflower plants using a mini settling tower supporting an inverted water agar plate containing leaf disks with P. xanthii telia (2) obtained from either sunflower or burweed. Plants covered with a settling tower were kept in the dark in a dew chamber for 48 h. Microscopic examination of inoculum from both sources confirmed germination of teliospores and formation of basidiospores. Test entries had similar disease reactions regardless of whether inocula were collected from H. annuus or X. strumarium. Fleck symptoms were visible on the hybrids SNK 77 and Orit 5 days after placing plants in the dew chamber. On these entries, telia gradually increased in size, indicating susceptibility to P. xanthii. Furthermore, those leaves receiving high inoculum dosages were desiccated 11 days after inoculation. Hybrids Hysun 333 and CRN 1470 and the male sterility maintainer lines DC 4 and RK 74 produced resistant reactions. Hyperplasia of stem and petiole tissue was observed on certain Orit, DC 4, and CRN 1470 plants. Susceptibility of burweed, a common and widespread annual weed in southern Africa (1), to a sunflower isolate of P. xanthii, was also confirmed. Depending on the formation of teliospores, the identity of the causal organism was verified in all inoculation studies. P. xanthii damage on sunflower has not been reported from South Africa. However, the disease has now become economically important as severe foliar lesions rendered plants unacceptable for the ornamental sunflower market.

First Report of Fusarium sambucinum, F. oxysporum, and F. subglutinans Associated with Stem Decay of Amaranthus hybridus in South Africa.

Amaranthus hybridus (common name: amaranth) is a fast-growing crop with nutritious leaves and seeds that is cultivated in semi-arid regions throughout the world. In South Africa, cultivation of this crop as a leafy vegetable is increasing. In autumn 1997, extensive tissue discoloration and decay were observed in branches, stems, and root collars of mature A. hybridus in Bloemfontein, Free State Province. Symptoms included discolored phloem, xylem, and pith, black cankers, and weakened stems prone to wind breakage. Examination of these tissues revealed larval galleries of the pigweed weevil (Hypolixus haerens), the main insect pest of A. hybridus in South Africa (1). Six-month-old A. hybridus stems were split and small samples of discolored tissue adjacent to the larval galleries of each stem and the associated larvae were placed aseptically on corn-meal agar containing streptomycin and incubated for 4 to 7 days. The seven fungi most frequently isolated from discolored stem tissues (n = 166) were Fusarium subglutinans (46%), a Phomopsis sp. (11%), Alternaria alternata (10%), F. oxysporum (9%), F. solani (5%), a Phoma sp. (5%), and F. sambucinum (4%). The nine fungi most frequently isolated from larvae (n = 90) were F. subglutinans (46%), F. solani (8%), F. equiseti (8%), F. oxysporum (7%), A. alternata (6%), a Phomopsis sp. (4%), F. proliferatum (3%), F. sambucinum (2%), and a Phoma sp. (2%). Stems of greenhouse-grown A. hybridus were inoculated with the seven most common species isolated from the discolored stem tissues. One isolate of each species was used. Inoculations involved wounding stems by removing approximately 36 mm2 of the epidermis 5 cm above the soil, placing a colonized water agar plug on the wound, and wrapping Parafilm around the stems at the wound site. Wounded and nonwounded (untreated) controls were also included. A noncolonized water agar plug was applied to wounded controls but not to nonwounded controls. Ten plants per isolate and 10 wounded and nonwounded control plants were used in each of two separate trials (180 total plants). Treatments were assigned randomly. Four weeks after inoculation, canker lengths were measured and stem sections were surface disinfected and transferred to water agar plates. The presence of the fungi was confirmed after 20 days. Only F. sambucinum, F. oxysporum, and F. subglutinans caused cankers with frequencies of 100, 100, and 65% (n = 20), and mean lesion lengths of 30, 26, and 10 mm, respectively. Lesions were never observed on either of the controls. Discoloration and cankers were similar to that observed in the field. F. sambucinum, F. oxysporum, and F. subglutinans were recovered from 65, 50, and 60% of the tissues, respectively, and none of the Fusarium spp. were recovered from the control treatments (n = 20 for all). In artificial inoculations, these species can act as pathogens independent of the pigweed weevil and are likely the cause of the discoloration, decay, and cankers observed in branches, stems, and root collars of mature A. hybridus. However, there are no prior reports of a Fusarium sp. causing disease on A. hybridus, and H. haerens larvae were observed in all symptomatic stems in the field. Further studies are needed to determine the potential for significant disease loss associated with this insect-fungal association and the potential role of these fungi in further weakening Amaranthus stems that are colonized by H. haerens. Reference: (1) S. vdM. Louw et al. Afr. Crop Sci. J. 3:93, 1995.