Agar Discs Research Articles

First Report of Sclerotinia sclerotiorum Infection on Cuphea.

Species of the genus Cuphea (family Lythraceae) are being developed as potential domestic sources of medium length fatty acids (lauric and capric) for use in industrial lubricants and detergents. During September 2004, patches of dead plants were observed in test plots of Cuphea sp. cv. PSR-23 (1) (Cuphea viscosissima Jacq. × C. lanceolata W.T. Aiton) near Morris, MN and Prosper, ND, approximately 200 km apart. Seed yield in the diseased Morris field was 78 kg/ha compared with 516 kg/ha in nearby, nonaffected fields of the same variety, for an 85% yield reduction. Stems were split open to reveal long, cylindrical sclerotia as much as 8 mm long. Isolations from diseased stem tissue and sclerotia were identified as Sclerotinia sclerotiorum (Lib.) de Bary and produced typical sized sclerotia (4 to 6 mm in diameter) after 7 days growth on potato dextrose agar (PDA). Cuphea PSR-23 plants were grown in the greenhouse in individual pots for 5 weeks and then inoculated. Three inoculation methods were used. For the first method, ascospores of a sunflower isolate of S. sclerotiorum were sprayed onto blooming flowers and foliage at a rate of 5,000 spores per ml. The inoculated plants were kept in a dark, 18°C mist chamber for 48 h and then returned to a greenhouse maintained at 24/20°C, day/night temperatures. All 20 inoculated plants were visibly colonized by Sclerotinia sp. after 3 days, and all plants were dead by 7 days. The second inoculation used the petiole inoculation technique employed by canola researchers (2). The blade from the third leaf was excised and a micropipette tip containing an agar disk of mycelia of the Cuphea isolate was placed over the cut end of the petiole. Five days after inoculation, all 30 inoculated plants were dead, while none of the 10 control plants (using sterile agar disks on the cut petiole) were affected. Isolations were made from diseased plants inoculated by all methods, and S. sclerotiorum colonies were observed on PDA medium with typical sclerotia from 4 to 6 mm in diameter. The third inoculation method tested root infection. S. sclerotiorum was grown on autoclaved proso millet (Panicum miliaceum L.) seed for 7 days, and 5 g of colonized millet seed was placed in a hole 6 cm from the base of a Cuphea plant, with one plant per 3.7 liter pot. Sunflower (Helianthus annuus L.; oilseed hybrid Cargill 270) plants served as inoculated controls. None of the 20 Cuphea plants were infected via soil inoculations compared with 70% of 30 sunflower plants that developed basal stalk rot and wilt within 2 weeks after inoculation. To our knowledge, this is the first report of S. sclerotiorum infection on Cuphea sp., and is believed to be the first report of infection on any genus within the Lythraceae (loosestrife family). With over 100 annual and perennial species in the genus Cuphea, the possibility of Sclerotinia spp. resistance needs to be investigated to further develop this potential oilseed crop.

Frequency and Pathogenicity of Dematiaceous Hyphomycetes on Annual Ryegrass Overseeded on Bermudagrass in Mississippi.

Fungal diseases of annual ryegrass (Lolium multiflorum Lam.), a cool-season species grown for forage and turf in the southeastern United States, and bermudagrass (Cynodon dactylon (L.) Pers.), a warm-season perennial, usually are considered distinct. In May 2002 and 2004, symptoms of leaf and stem necrosis were observed simultaneously in forage bermudagrass and overseeded annual ryegrass on a swine waste application site in Mississippi. Sporulation by nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum was observed on symptomatic leaves of ryegrass that were surface disinfested and plated on agar each year, and axenic cultures were established by spore transfers. These isolations represent four new worldwide and two new North American records of occurrence of fungal pathogens on ryegrass, and three new records for the southeastern United States and Mississippi. Bipolaris cynodontis, a common bermudagrass pathogen, and Drechslera dictyoides were observed most frequently on ryegrass during both years. In excised leaves of ryegrass inoculated with infested agar discs, most isolates of B. cynodontis from ryegrass and bermudagrass were equally virulent and caused necrosis equal to or greater than that caused by D. dictyoides, an established ryegrass pathogen. Isolates of B. cynodontis from both hosts also caused similar symptoms in foliage of ryegrass and bermudagrass following spore inoculations. Pathogenicity to ryegrass of five other species of dematiaceous hyphomycetes that represented new worldwide or North American records of occurrence also was demonstrated by foliar inoculations. All pathogens were reisolated from symptomatic tissues and grown in axenic culture for fulfillment of Koch's postulates. Results demonstrate that most of the same species of dematiaceous hyphomycetes infect both forage ryegrass and bermudagrass in Mississippi and indicate that B. cynodontis, in particular, may be a common and virulent pathogen of ryegrass in the southeastern United States.

First Report of Infection of Maiden-Hair Fern (Adiantum jordanii and A. aleuticum) by Phytophthora ramorum in California.

During July 2005, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from nine native Adiantum jordanii plants growing at two forest sites (Samuel P. Taylor State Park, Marin County and Peachland Road, Mendocino County) and from seven A. aleuticum plants at one forest site (Peachland Road) in California. At both locations, symptomatic plants were distributed close to rivers and roads and in association with infected bay laurel trees (Umbellularia californica), toyon (Heteromeles arbutifolia), and tanoaks (Lithocarpus densiflorus). Symptomatic leaflets showed brown spots that sometimes coalesced, killing entire leaves, but the disease did not appear to be fatal to the ferns. Necrotic tissues were plated on PARP and maintained in the dark at 18°C for 1 to 2 weeks. Isolates were identified as P. ramorum on the basis of colony morphology, the presence of chlamydospores and caducous, semipapillate sporangia, and the internal transcribed spacer (ITS) rDNA sequences (1,2). The P. ramorum isolates, Pr-419 from A. jordanii and Pr-422 from A. aleuticum, have been deposited in the American Type Culture Collection (ATCC MYA-3677 and MYA-3679, respectively) and a region of the ITS rDNA sequence deposited in the NCBI database (GenBank Accession No. DQ173082 and DQ219821, respectively). To test the pathogenicity, the tips of freshly detached leaves of A. jordanii and A. aleuticum were dipped into a solution of 1 × 103 zoospores per ml of Pr-419 and Pr-422 for 1 min. The wounded end of the leaves was not exposed to the inoculum. The zoospores were produced by flooding agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar with sterile deionized water. After 3 days of incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then at room temperature for 60 min. For each Adiantum species and P. ramorum isolate, 15 leaves collected from five potted nursery plants were tested. Control leaves were dipped in sterile deionized water. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 9 days. Brown lesions similar to those seen in the forest developed on approximately 60 and 33% of the A. jordanii and A. aleuticum leaves, respectively, inoculated with Pr-419 and on approximately 73 and 40% of the leaves inoculated with Pr-422. Under these experimental conditions, A. aleuticum appeared to be slightly more susceptible than the A. jordanii, with a necrotic leaf area of approximately 38% compared with 20%. The pathogen was reisolated on PARP after surface sterilization from all symptomatic leaves. Control leaves did not develop symptoms and P. ramorum was not recovered. A. jordanii and A. aleuticum have already been listed as associated hosts for P. ramorum on the APHIS (USDA Animal and Plant Health Inspection Service) website ( http://www.aphis.usda.gov/ ). To our knowledge, this is the first report of ferns as natural hosts of P. ramorum.

Guava Fruit Anthracnose and the Effects on its Nutritional and Market Values in Ibadan, Nigeria

Guava Fruit Anthracnose and the Effects on its Nutritional and Market Values in Ibadan, Nigeria

First Report of Root Rot of Cocoyam Caused by Pythium myriotylum in Sri Lanka

Root rot of cocoyam (Xanthosoma sagittifolium L. Schott) caused by Pythium myriotylum Drechsler is a major disease of this crop in Africa (1,2) but is unreported from other regions of the world. During September 1999, commercially grown cocoyam (cv. Ratu-kiri-ala) in Gampaha (7°05'N, 80°00'E), Sri Lanka suffered from severe root rot. Initial symptoms were water-soaked lesions at the root tips that gradually enlarged to rot the entire root system and tuber. Wilting and yellowing of leaves were observed in advanced stages of disease. A Pythium sp. was regularly isolated from the affected roots and an isolate, SC5, was identified as P. myriotylum on the basis of morphology and the internal transcribed spacer (ITS) rDNA sequence. Characteristics of isolate SC5, grown on a grass-leaf water culture (3) were main hyphae up to 8.5 μm wide, oogonia terminal or intercalary (22.5 to 33.8 μm in diameter), antheridia diclinous occasionally monoclinous, one to eight per oogonium, stalks branched, often more or less loosely enveloping the oogonium, antheridium clavate or crook-necked, making apical contact with the oogonium, breadth of antheridium 2.5 to 7.0 μm, oospores aplerotic (17.0 to 22.5 μm in diameter), oospore wall 0.8 to 2.0 μm in thickness, sporangia terminal or intercalary, filamentous, inflated lobulate, and digitate, of variable length, breadth of sporangia 7.0 to 17.5 μm, formed in water; zoospores formed at 25°C, and diameter of encysted zoospores 10.0 to 12.5 μm. Cardinal temperatures on potato carrot agar 8°C minimum, 34°C optimum, and 37°C maximum with daily radial growth rate for 34°C at 32.8 mm. The ITS rDNA sequence of the isolate matched the sequences of P. myriotylum in GenBank (Accession Nos. AB095051 and AF452156) and isolate CBS254.70 used for the species description by van der Plaats-Niterink (3). The sequence of SC5 has been deposited in GenBank, Accession No. DQ102701. Pathogenicity tests used potted cocoyam plants (20 cm high), planted in an autoclaved potting mix. Four agar disks (8 mm in diameter) of isolate SC5 grown at 25°C for 48 h on potato dextrose agar was mashed and injected at a depth of 2 to 3 cm in the soil around the roots. Inoculated plants were placed in transparent plastic bags and kept for 7 days in a growth chamber maintained at 24 to 26°C with continuous light (52 to 98 μmol m-2·s-1). The experiment was carried out twice with three replications for each test. Dark brown rotting on roots and wilting of leaves were observed in 7 days after the inoculation. P. myriotylum was reisolated from diseased tissues and found to be morphologically identical to the original isolate SC5. Noninoculated control plants remained healthy. On the basis of the symptoms, morphological and molecular characteristics and confirmation of pathogenicity, P. myriotylum is the causal agent of root rot of cocoyam. To our knowledge, this is the first report of P. myriotylum causing root rot of cocoyam in Sri Lanka.

Anti‐angiogenic effects and regression of localized murine AML produced by anti‐VEGF and anti‐Flk‐1 antibodies

Reducing the blood supply of tumors is one modality to combat cancer. The objective of this study was to evaluate such an approach in the treatment of localized murine AML (acute myelogenous leukemia). For this purpose we designed an experimental model in which leukemic cells were embedded in 1% agar discs before subcutaneous implantation in C57Bl female mice. The C-1498 AML cell line (Frederick Inst., NCI, MD, USA) was used. Thirty experimental mice received on alternate days injections of 5 x 2.5 microg anti-VEGF (vascular endothelial growth factor) and 5 x 2.5 microg anti-Flk-1 (VEGFR2) antibodies to the site of cell implantation over a period of 10 d. Fifteen control mice received daily PBS injections. All mice were sacrificed 16 d after AML implantation. Of the 30 experimental animals, macroscopic examination showed in 21 animals (70%) small sized, pale tumors (0.5 g); in six mice (20%) the tumors were replaced completely by necrotic tissue, while in three mice (10%), there were large (2.5 g), highly vascularized tumors. In all 15 control mice large highly vascularized tumors were seen. A separate group of mice was studied for total survival following AML implantation. While 12 mice in the control group not treated with antibodies survived for 16 d post-implantation, survival was prolonged in 15 antibody treated mice by approximate 30 d to a total survival time of 48 d. Tumor specimens were processed for histology, immunohistochemistry (IHC) for CD31 endothelial cell antigen, and tube-like formation assay. The small, pale tumors of antibody treated animals consisted of degenerate hyaline material with remnant nests of leukemic cells, whereas large tumors showed sheets of leukemic cells and numerous blood vessels. Specimens processed for CD31 antigen showed scarce or absence of blood vessels in the small, pale tumors in contrast to intensive staining from a rich network of blood vessels in the large, highly vascularized tumors. Tube-like formation assays disclosed rudimentary Grade 1 endothelial cell tubes in the small, pale tumors as opposed to polygonal Grade 4 tube formation in control animals. In conclusion, this murine model of localized AML allows assessment of anti-angiogenic tumor regression. Anti-angiogenic antibodies against VEGF and Flk-1 have therapeutic effects in murine AML.

First Report of Rhizoctonia solani AG-4 and AG-2-2 on Lupinus angustifolius in Canada.

Narrow-leaved lupine (Lupinus angustifolius L.) is grown as a grain legume crop in Australia and Europe where it is used as feedstock in the livestock and aquaculture industries. During July 2003, a stem rot disease was observed in narrow-leaved lupine (cv. Arabella) plants in a research plot at the Crop Diversification Centre North (CDCN), Edmonton, Alberta, Canada. The disease was also found on cv. Rose at the CDCN and cv. Arabella in experimental fields near Devon, Ellerslie, and Westlock, Alberta during the late spring of 2004. Diseased plants showed dark brown-to-black stems with sunken and constricted lesions at the soil level. Young leaves became shrunken and twisted and seedlings collapsed. Rhizoctonia solani was consistently isolated from lesions on taproots and basal stems of diseased plants. Colonies of cream-colored mycelia grew close to the surface of potato dextrose agar (PDA). Most sclerotia formed inside the medium. Agar disks (1 cm in diameter) of isolates LP-11Bb, LP-24C, and LP-25C were attached to the opposite sides of basal stems (180° apart) of 1-month-old lupine seedlings (cv. Arabella). Inoculated plants were incubated for 2 days in black plastic bags under a greenhouse bench at approximately 20°C. All isolates caused brown lesions on the lower stems (extending up to 7 cm above ground level), girdling, and root rot. Plants wilted within 7 to 10 days after inoculation, and aerial mycelia appeared on the basal stems. R. solani was reisolated from the infected crown tissues using PDA to complete Koch's postulates. The isolates were paired with AG tester strains of R. solani. Isolates LP-11Bb and LP-24C were identified as AG-4 while isolate LP-25C was identified as AG-2-2. In another trial, eight isolates of R. solani (unknown AG types) were tested for virulence on L. angustifolius cv. Arabella using the inoculation method described above. All isolates were pathogenic, and disease severity that was based on a 0 to 4 scale ranged from 2.7 to 3.2. The most virulent strain was LP-24C, which caused a 77% loss in fresh weight compared with the noninoculated control plants. R. solani AG-8 is associated with Rhizoctonia disease of lupine in Australia (1) and also causes bare patch disease of wheat. To our knowledge, this is the first report of R. solani on lupine in Canada. This disease could have a significant impact on the commercial production of lupine in Alberta. Reference: (1) M. W. Sweetingham et al. Pages 466-486 in: Advances in Lupin Disease Management in Australia. Proc. Int. Lupin Conf., 8th. G. D. Hill, ed. International Lupin Association, Canterbury, New Zealand, 1999.

Responses of common SE Australian herbivores to three suspected invasive Caulerpa spp.

We sought to determine whether common intertidal and shallow subtidal zone grazers would consume extracts or fronds of three invasive Caulerpa spp., all of which are now resident in southern New South Wales, Australia. We examined the responses of herbivorous fishes, echinoderms and molluscs to C. filiformis. A subset of these organisms was tested with extracts of C. scalpelliformis and C. taxifolia. Polar (seawater) extracts of C. filiformis deterred a single herbivore, Aplysia sydneyensis, but confirmed that the biological activity reported from some Caulerpa spp. is not restricted to the lipophilic fractions. The large turbinid Turbo torquatus was deterred by an ethanol extract of C. filiformis, while the small congener T. undulatus demonstrated a significant preference for palatable agar discs containing ethanol extracts of C. filiformis. However, when T. undulatus were offered a choice of fronds from five algal species in the laboratory, they readily consumed Ulva spp. and Sargassum sp., showing the lowest preference for C. filiformis. Solvent extracts of C. scalpelliformis and C. taxifolia did not significantly deter any grazers. However, the overall trend was for reduced consumption of discs containing solvent extracts of these seaweeds. Indeed, for the large urchin Centrostephanus rodgersii and in the fish trials these effects were very near significant (P<0.06). We conclude that common herbivores associated with hard substrata are highly unlikely to intercede in the spread or control of these invasive algae.

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×109 colony-forming units per milliliter or higher and pathogen spore suspensions of 5×104 spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.

Biological Control of Pythium Damping-off of Bush Okra Using Rhizosphere Strains of Pseudomonas fluorescens

A severe damping-off disease of bush okra caused by Pythium aphanidermatum, was diagnosed in plastic houses in Der Attia village, 15 km southwest of El-Minia city, Egypt, during the winter of 2001. Bush okra seedlings showed low emergence with bare patches inside the plastic houses. Seedlings that escaped pre-emergence damping-off showed poor growth, stunting and eventually collapsed. Examination of the infected tissues confirmed only Pythium aphanidermatum, showing its typical intercalary antheridia, and lobulate zoosporangia. P aphanidermatum was shown to be pathogenic on bush okra under pot and field experiments. Bacteria making inhibition zones against the damping-off fungus P. aphanidermatum were selected. Agar discs from rhizosphere soil of bush okra containing colonies were transferred onto agar plate culture of P. aphanidermatum. After 2 days of incubation, colonies producing clear zones of non-Pythium growth were readily detected. The two bacteria with the largest inhibition zones were identified as Pseudomonas fluorescent. Bush okra emergence (%) in both pot and plastic houses experiments indicated that disease control could be obtained by applying P. fluorescent to the soil or coating the bacteria to the bush okra seeds before sowing. In the plastic houses, application of the bacteria onto Pythium-infested soil and sowing bush okra seeds dressed with bacteria gave 100% emergence. In addition, This was the first reported disease of bush okra by this oomycete in Egypt.

Phytophthora cinnamomi

<i>Phytophthora cinnamomi</i>

First Report of Phytophthora nicotianae on Skimmia japonica in Italy.

Skimmia japonica, an evergreen flowering shrub, is becoming increasingly popular as a potted ornamental plant in northern Italy and represents 5% of acidophilous plant production; cv. Rubella accounts for 99% of production. During the spring of 2003, in many commercial nurseries located in northwestern Italy, plants of S. japonica cv. Rubella showed extensive chlorosis and root rot, and diseased plants eventually wilted and died without dropping leaves. The disease was widespread and severe, and in some nurseries, 40% of plants were affected. A Phytophthora-like organism was isolated consistently from infected lower stem and root pieces of S. japonica that had been disinfested for 1 min in 1% NaOCl and plated on a medium selective for oomycetes (2). The pathogen was identified based on morphological and physiological features as Phytophthora nicotianae (= P. parasitica [1]). The sporangia produced on V8 medium were ± spherical to obpyriform, obturbinate, papillate, and measured 33 to 94 × 25 to 48 μm (average 56.4 × 36.8 μm). Papillae measured 3.5 to 19 μm (average 7.8 μm). Chlamydospores were spherical with a diameter ranging from 26 to 32 μm (average 29.2 μm). Pathogenicity of four isolates obtained from infected plants was confirmed by inoculating 9-month-old plants of S. japonica cv. Rubella grown in 1-liter pots containing a substrate based on sphagnum peatmoss, pine bark, and clay (70-20-10% vol/vol/vol). Inocula, which consisted of 90-mm-diameter V8 agar disks per pot containing mycelium of each isolate, were introduced and mixed into the substrate in all pots before transplanting. One plant was transplanted into each pot and served as a replicate, and noninoculated plants served as controls. Eight replicates were used for each isolate and the control treatment, and the trial was repeated. All plants were kept outside at temperatures ranging from 16 to 38°C (average temperature 27°C). Inoculated plants developed symptoms of chlorosis, root rot, and wilt within 20 days, while control plants remained symptomless. P. nicotianae consistently was isolated from inoculated plants. Previously, P. nicotianae has been reported on S. japonica in Poland (3). To our knowledge, this is the first report of P. nicotianae on S. japonica in Italy.

POST-HARVEST FUNGAL DISEASES OF PAWPAW (&lt;i&gt;CARICA PAPAYA&lt;/i&gt; L.) FRUITS AND SEEDS IN NIGERIA

Post-harvest fungal diseases of pawpaw ( Carica papaya L.) fruits sold in Mile 3 Market, Port Harcourt were investigated bi-weekly for sixteen weeks using the Standard Blotter Method. The following fungi were isolated from the tissues of diseased fruits: Fusarium solani, Phoma carica-papaya Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Cladosporium herbarum, Colletotrichum dematium, Fusarium moniliforme, Phomopsis carica-papaya, Penicillium sp., and Rhizopus stolonifer . Seeds of diseased fruits were also tested for health using the Standard Blotter Method and all the fungi isolated from the fruit tissues were found to be seed-borne except F. moniliforme . Pathogenicity test of all the fungi isolated from ripe fruit tissues and seeds were carried out on mature green pawpaw fruits and they were found to be pathogenic. Rhizopus stolonifer and F. solani caused the greatest rot on the fruits. Agar discs (3.0mm, diameter) of these fungi were subjected to hot water treatment at varying periods of time, (40°C for 30 mins., 50°C for 20 mins., and 60°C for 10 mins.) to determine the efficacy of the treatments on the pathogens. The hot water treatments could not eliminate the pathogens; instead their lineal growths on PDA medium were just inhibited, when compared with their controls. A first report of seed-borne fungi of pawpaw and their pathogenicity is provided. KEY WORDS: Pawpaw fruits, seed-borne fungi, pathosenicity test, hot water treatment. Global Jnl Pure & Applied Sciences Vol.10(1) 2004: 69-73

Effects of Cryptococcus laurentii (Kufferath) Skinner in combination with sodium bicarbonate on biocontrol of postharvest green mold decay of citrus fruit

The potential of using Cryptococcus laurentii alone or in combination with sodium bicarbonate solution for control of Penicillium digitatum (green mold) on oranges was investigated. Agar disks of C. laurentii NYDA cultures placed on PDA plates seeded with pathogen did not inhibit the growth of P. digitatum. Spore germination of P. digitatum in PDB was significantly controlled in the presence of living C. laurentii cell suspensions. Cryptococcus laurentii significantly controlled green mold on oranges after challenge with 5 × 104 spores/ml of P. digitatum. The higher the concentrations of the antagonist, the lower the disease incidence regardless of whether the fruit was stored at 20°C for 5 days or 2°C for 50 days. The efficacy of C. laurentii for control of green mold was improved when combined with sodium bicarbonate. The combination of sodium bicarbonate and C. laurentii could be an alternative to fungicides for control of postharvest green mold disease on citrus fruits.

Spore deposition of wood‐decaying fungi: importance of landscape composition

The abundance of many species of wood‐decaying fungi has decreased dramatically in Swedish boreal forests over the last century. Therefore, we have investigated the relationship between the spore dispersal of wood‐decaying fungi and two key features of landscape composition, namely the amount and age of old forest stands at different spatial scales. Spore deposition was monitored in two regions, using sampling methods based on recording the dikaryotisation of monokaryotic mycelia on nutrient agar and wood discs. The studied species, all mainly confined to Norway spruce in the boreal forest zone, were Fomitopsis pinicola,Fomitopsis rosea, Gloeoporus taxicola, Phlebia centrifuga and Trichaptum laricinum. The study of forestry intensity showed for both regions, that the spore deposition for F. pinicola, F. rosea and G. taxicola was higher in circular plots (radius 2‐km) with a high proportion of old Norway spruce forest (&gt;80 yr) than in plots with a lower proportion of old forest. Analysis of the variation in spore deposition of F. rosea and P. centrifuga in relation to the proportion of old spruce forest within 1, 2 and 3 km of the spore sampling point showed that the proportion of old forest within a 3‐km radius explained more of the variation than the proportions within 1‐ and 2‐km radii. In addition, the proportion of forest older than 140 yr explained more of the variation than the proportion of younger forests. Thus, the results show that the spore deposition of the studied species strongly depends on the landscape composition at both regional and local scales. Further, the spore deposition at the local scale was best explained by the proportion of &gt;140 yr old spruce forest, which exceeds the common harvest rotation period.

Double-disk synergy test positivity in Stenotrophomonas maltophilia clinical strains.

The double-disk synergy test (DDST) using Mueller-Hinton agar and antibiotic disks with centrally positioned disks of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam and, at a center-to-center distance of 25-30 mm, 2-4 disks with 10 various beta-lactam antibiotics per one plate was performed in 58 clinical isolates of Stenotrophomonas maltophilia to determine the effectivity of 3 beta-lactamase inhibitors. When tested with clavulanate as the central beta-lactamase inhibitor synergic action on tested strains was the most frequent with aztreonam (81.0% of strains), cefoperazone (63.8%), and cefepime (60.3%). With sulbactam the synergic action, i.e. DDST positivity, was high in the case of cefoperazone (15.5%), ampicillin, aztreonam and piperacillin (8.6% each); with tazobactam it was the most frequent with aztreonam (53.4%), cefoperazone (44.8%) and cefepime (37.9%). No synergy was demonstrated after application of meropenem regardless of the kind of beta-lactamase inhibitor used. In 58 strains of S. maltophilia, 55 different profiles of DDST positivity were found. The results confirm that clavulanate is the most effective inhibitor of S. maltophilia beta-lactamases. The utilization of DDST (performed in the recommended way) for the typization of strains Stenotrophomonas species and for the estimation of potential effectiveness combinations of beta-lactams with beta-lactamase inhibitors for the therapy of stenotrophomonade infections was suggested.

Effects of Cryptococcus laurentii (Kufferath) Skinner on biocontrol of postharvest decay of arbutus berries

Cryptococcus laurentii was tested as a biocontrol agent for reducing natural decay of arbutus berries caused by Penicillium citrinum and Verticicladiella abielina in semi-commercial postharvest trials. Three different preparations of C. laurentii were compared for antagonistic efficiency. Washed C. laurentii cells provided better protection against decay than yeast cultured in broth without washing while the culture supernatant free of yeast cells provided no protection. The protection provided by the washed yeast cells was dose-dependent. Cryptococcus laurentii was also effective in controlling decay at low temperature (4°C). The efficacy of C. laurentii was enhanced by the addition of 2% CaCl2. Agar disks of C. laurentii NYDA cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. citrinum or V. abielina. Spore germination of the pathogens in potato dextrose broth was strongly inhibited in the presence of active cell suspensions.

Anastomosis Grouping of Rhizoctonia solani Associated with Black Scurf and Stem Canker of Potato in South Africa.

Rhizoctonia disease (black scurf of tubers and stem canker) of potato (Solanum tuberosum L.) caused by Rhizoctonia solani Kühn was first recorded in South Africa in 1918 (3). Although the sclerotial form on tubers is one of the most common potato diseases in the country, it is not known which anastomosis groups (AGs) of R. solani are involved. Between 1999 and 2001, R. solani was isolated from 28 plant and 56 soil samples collected in 7 (Eastern Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape, and Sandveld) of the 14 potato-production regions of South Africa and screened for hyphal anastomosis with tester strains of R. solani AG-1 to AG-10 according to Carling et al. (1). Of the 411 isolates from tubers with black scurf symptoms, 408 were AG-3 and three were AG-5. Symptomless tubers yielded two AG-3 isolates and three AG-5 isolates. Of 39 isolates from symptomatic stems and roots, 32 were AG-3, five were AG-4, and two were AG-5. Of the 127 isolates obtained from soil, 86, 28, 7, 3, and 3 were AG-3, AG-4, AG-5, AG-7, and AG-8, respectively. More than one AG was isolated from five of the seven regions. Virulence of 40 isolates representative of the above AGs was determined in triplicate on sprouts growing from seed tubers of potato cultivar Up-to-Date in a sand/soil mixture as described by Carling and Leiner (2) but using cultures grown in cornmeal/sand instead of colonized agar disks as inoculum. Damage to sprouts (lesions, girdling, and death) was assessed after 28 days at 16 to 28°C according to the 0 to 4 rating scale (2). Chi-square analysis of the data indicated that AG-3 was the most virulent, with isolates from sclerotia on tubers and lesions on stems more aggressive than those from symptomless tubers or soil. AG-4 and AG-5 caused significantly less disease than AG-3, but none of the AG-7 and AG-8 isolates showed any virulence to potato sprouts.

First report of orchid (Orchis laxiflora) as a host of Sclerotinia minor, discovered in Turkey

In Erzurum, Turkey, during June–July, 2002, Orchis laxiflora was affected by a previously undescribed disease. Initial symptoms consisted of a soft, watery rot at the crown and stem. Diseased tissue turned black, extending along the stem, before plants wilted and collapsed. The pathogen was isolated from affected stem sections, surface-disinfected for 1 min in a solution of NaOCl, producing 0·5% available Cl2, plated on potato dextrose agar (PDA) and incubated at 25°C. The organism isolated produced white mycelia and small (0·5–2·5 mm) black sclerotia characteristic of Sclerotinia minor (Pratt, 1993). This pathogen was consistently isolated from these plants. Symptoms were reproduced in the glasshouse by inoculating stems of 4-month-old plants with mycelial plugs (6 mm diameter) from 5-day-old PDA cultures. Inoculated plants were enclosed in transparent plastic bags for 3 days. Control plants were treated similarly, except that the agar disks did not contain the fungus. All plants were incubated in a glasshouse at 21–23°C with a 12-h photoperiod. After 2 weeks, inoculated O. laxiflora plants wilted and collapsed. S. minor was reisolated from necrotic crown and stem tissues. No disease symptoms were observed on uninoculated plants. The experiment was repeated and results were similar to the first series of inoculations. Sclerotinia minor causes diseases on an extremely broad range of host plants worldwide. A recent listing of hosts by Melzer et al. (1997) included 21 families, 66 genera and 94 species. This is the first report of O. laxiflora as a host of S. minor.

First Report of a Labyrinthula sp. Causing Rapid Blight Disease of Rough Bluegrass and Perennial Ryegrass.

A Labyrinthula sp. was isolated from symptomatic rough bluegrass (Poa trivialis L.) and perennial ryegrass (Lolium perenne L.) from a golf course in Arizona. Initial symptoms were a water-soaked appearance and rapid collapse of small patches of turf foliage. The affected turf died, and patches coalesced to form large dead areas after several weeks. The symptoms were those of the disease recently termed "rapid blight" for which the causal agent has not been identified (1). Rapid blight was first observed in southern California in 1995 and has become increasingly problematic in 10 other states on several cool-season turfgrasses (1). In Arizona, it is associated with high salinity irrigation water. In microscopic examinations of symptomatic P. trivialis and L. perenne leaf tissue from November 2002 to February 2003, fusiform or spindle-shaped vegetative cells (4 to 5 × 15 to 20 μm) were observed in leaf cells. These cells are consistently associated with rapid blight (1) and are typical in size and shape of those described for Labyrinthula spp. (3,4). The fusiform cells were cultured in 1% horse serum water agar medium made with irrigation water (electrical conductivity [EC] = 3.5 to 4.0 dS/m) from a golf course in central Arizona with rapid blight. The cells readily formed colonies on this medium and exhibited gliding motility along a network of hyaline slime filaments as previously described for the genus Labyrinthula (3,4). Koch's postulates were fulfilled by inoculating P. trivialis and L. perenne seedlings with Labyrinthula sp. isolated from naturally infested P. trivialis in two experiments. The grasses were started from seed and grown as a lawn in containers in the laboratory. Both experiments were repeated once. In the first experiment, infested autoclaved leaf pieces of P. trivialis were used as inoculum. Inoculated leaf pieces were placed within each of several bundles of 4 to 6 leaves and held loosely in place with a 0.5-cm wide ring of tygon tubing. Seedlings were irrigated with sterilized irrigation water from the golf course (EC = 4.0 dS/m). In the second experiment, agar discs from Labyrinthula sp. colonies on 1% horse serum agar were used as inoculum by placing the agar discs in contact with leaves. Seedlings were irrigated with sterile tap water adjusted to 4.0 dS/m using synthetic sea salt (Instant Ocean, Aquarium Systems, Inc., Mentor, OH) Leaf tissue of all inoculated seedlings became water soaked within 3 to 7 days and collapsed within 10 days in both experiments. Fusiform cells were observed in inoculated leaf tissue cells, and the Labyrinthula sp. was reisolated from 100% of selected symptomatic seedlings. Control seedlings treated with noninfested leaf pieces or sterile agar pieces did not develop symptoms, and no fusiform cells were isolated from the leaf tissue. Labyrinthula spp. are usually associated with marine systems (3). Labyrinthula zosterae D. Porter & Muehlst. has been identified as the causal agent in a marine grass wasting disease (2), but to our knowledge, no Labyrinthula spp. have been described as pathogens of terrestrial plants.