In vitro and In vivo Effects of Some Fungicides against the Chickpea Blight Pathogen, Ascochyta rabiei

The effects of various fungicides on mycelial growth and spore germination of Ascochyta rabiei were determined by incorporating them into potato dextrose agar and measuring colony diameter and observing colony growth and spore germination at 20 ± 2°C. Eight fungicides prevented spore germination of the pathogen at concentrations of 0.125–2 μg/ml, three hindered mycelial growth at 2–4 μg/ml and seven failed to inhibit mycelial growth even at 128 μg/ml. The reference fungicide for the pathogen, chlorothalonil, stopped conidial germination at low rates but did not prevent mycelial growth at 128 μg/ml. Thirteen fungicides were tested against seed infections of the pathogen, and benomyl + thiram, carbendazim and carbendazim + chlorothalonil seed treatments gave more than 85% inhibition on both vacuum‐infiltrated and naturally infected seeds. Coating the seeds with polymers did not increase the effectiveness of fungicides. Three fungicides; (azoxystrobin, chlorothalonil and mancozeb), gave the highest protection in the field but protection decreased with increased inoculum pressure. Addition of humic acid to fungicide suspensions did not affect their performance.     

Effects of Selected Fungicides and the Timing of Fungicide Application on Beauveria bassiana-Induced Mortality of the Colorado Potato Beetle (Coleoptera: Chrysomelidae)

Four fungicides used for controlling foliar diseases of potato (Solanum tuberosum) were evaluated under field and laboratory conditions for their effects on the infectivity and sporulation of Beauveria bassiana when used as a control for the Colorado potato beetle, Leptinotarsa decemlineata (CPB). We investigated the direct effects of fungicides on B. bassiana-induced CPB mortality and the effect of time between fungicide and B. bassiana application. Effects of fungicide on conidial survival in the soil and on foliage were examined in the field. Significantly more larval mortality was observed when larvae were sprayed with B. bassiana than with the water control. Fungicide had no significant effect on larval mortality in the field. In the laboratory, survival of larvae was significantly lower among larvae fed fungicide-treated foliage. B. bassiana-induced mortality in the laboratory was observed only when larvae were fed foliage treated with copper hydroxide or water. Larvae fed mancozeb- or chlorothalonil-treated foliage experienced high mortality regardless of B. bassiana treatment. While there was no significant effect of fungicide on B. bassiana sporulation on cadavers in the field, a pattern emerged that indicated higher proportions of cadavers producing conidia in plots sprayed with water or copper hydroxide than in plots sprayed with chlorothalonil or mancozeb. Survival of B. bassiana conidia in the soil and on foliage was significantly greater in plots treated with copper hydroxide or water than in plots treated with mancozeb or chlorothalonil. Fungicides such as copper hydroxide may be less deleterious to the fungus than mancozeb and chlorothalonil.     

Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue

The effect of mechanical wounding or foliar diseases caused by Sclerotinia homoeocarpa or Rhizoctonia solani on the epiphytic yeast communities on creeping bentgrass and tall fescue were determined by leaf washing and dilution plating. Total yeast communities on healthy bentgrass and tall fescue leaves ranged from 7.9 x 103 to 1.4 x 105 CFU.cm-2 and from 2.4 x 103 to 1.6 x 104 CFU.cm-2, respectively. Mechanically wounded leaves (1 of 2 trials) and leaves with disease lesions (11 of 12 trials) supported significantly larger communities of phylloplane yeasts. Total yeast communities on S. homoeocarpa infected or R. solani infected bentgrass leaves were 3.6-10.2 times and 6.2-6.4 times larger, respectively, than the communities on healthy leaves. In general, healthy and diseased bentgrass leaves supported larger yeast communities than healthy or diseased tall fescue leaves. We categorized the majority of yeasts as white-pigmented species, including Cryptococcus laurentii, Cryptococcus flavus, Pseudozyma antarctica, Pseudozyma aphidis, and Pseudozyma parantarctica. The percentage of pink yeasts in the total yeast community ranged from 2.6% to 9.9% on healthy leaves and increased to 32.0%-44.7% on S. homoeocarpa infected leaves. Pink-pigmented yeasts included Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, and Sporidiobolus pararoseus. Foliar disease significantly affected community size and composition of epiphytic yeasts on bentgrass and tall fescue.     

Variable adhesion and diurnal population patterns of epiphytic yeasts on creeping bentgrass

Irrigation and an in vitro agitation assay were used to determine the percentage of the epiphytic yeast community (Cryptococcus, Pseudozyma, Rhodotorula, and Sporobolomyces) adhering to the phylloplane of creeping bentgrass (Agrostis palustris (Huds.) Pers.). Colony-forming units (cfu) of total epiphytic yeast populations (adherent and nonadherent cells) and of adherent populations (cells not removed by agitation) were determined by leaf washing and dilution plating. In an in vitro assay, 40.0% and 57.1% of the yeast adhered to the leaves, whereas, in initial field trials the percentage of adherent yeasts ranged from 40.0% to 71.9% of the total population. Adherent yeast cfu on leaves in the morning were significantly lower on bentgrass (8.0 x 103 to 3.1 x 104 cfu.cm(-2)) compared with total yeast cfu (1.4 x 104 to 4.7 x 104 cfu.cm(-2)) on the nonirrigated control. No differences in yeast populations were observed between irrigated and nonirrigated plots 2 h after the 0900 treatments. Yeast populations followed a diurnal pattern, with larger cfu recovered from bentgrass leaves in the morning and significantly lower populations recovered in the afternoon. At 1400 the adherent yeast were 83.1%-100% of the total yeast population recovered from the leaves. The relative adhesiveness of the epiphytic yeast community on bentgrass leaves is dynamic with nonadherent cells making up a larger percentage of the population in the mornings than the afternoons.     

The influence of fungicides on Arthrobotrys oligospora in simulated putting green soil

Plant‐parasitic nematodes are destructive pests in bentgrass putting greens. Few chemical or nonchemical approaches for nematode management exist. Studies were conducted to determine: the in vitro tolerance of the nematophagous fungus Arthrobotrys oligospora, to the fungicides chlorothalonil and myclobutanil used to manage diseases on putting greens; the concentration of fungicides obtained from simulated putting green soil; and the ability of the fungus to reduce populations of the ring nematode, Criconemella ornata. Both fungicides reduced in vitro hyphal growth and germination of conidia above 10 mg kg^‐1. Soil concentrations of chlorothalonil were less than 5 mg kg^‐1 and concentrations of myclobutanil were below detection limits. Nematode populations were not affected by A. oligospora in simulated greens but nematode populations were lowest in pots inoculated with A. oligospora and receiving fungicide treatments. Results of these studies indicate that applications of chlorothalonil and myclobutanil used to manage fungal diseases of bentgrass may not adversely affect A. oligospora; however, the fungus may not reduce nematode populations below desired thresholds.     

In vitro attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa

The ability of yeasts to attach to hyphae or conidia of phytopathogenic fungi has been speculated to contribute to biocontrol activity on plant surfaces. Attachment of phylloplane yeasts to Botrytis cinerea, Rhizoctonia solani, and Sclerotinia homoeocarpa was determined using in vitro attachment assays. Yeasts were incubated for 2 d on potato dextrose agar (PDA) prior to experimentation. A total of 292 yeasts cultured on PDA were screened for their ability to attach to conidia of B. cinerea; 260 isolates (89.1%) attached to conidia forming large aggregates of cells, and 22 isolates (7.5%) weakly attached to conidia with 1 or 2 yeast cells attached to a few conidia. Ten yeasts (3.4%), including 8 isolates of Cryptococcus laurentii, 1 isolate of Cryptococcus flavescens, and an unidentified species of Cryptococcus, failed to attach to conidia. All non-attaching yeasts produced copious extracellular polysaccharide (EPS) on PDA. Seventeen yeast isolates did not attach to hyphal fragments of B. cinerea, R. solani, and S. homoeocarpa after a 1 h incubation, but attachment was observed after 24 h. Culture medium, but not culture age, significantly affected the attachment of yeast cells to conidia of B. cinerea. The 10 yeast isolates that did not attach to conidia when grown on agar did attach to conidia (20%-57% of conidia with attached yeast cells) when cultured in liquid medium. Attachment of the biocontrol yeast Rhodotorula glutinis PM4 to conidia of B. cinerea was significantly greater at 1 x 10(7) yeast cells x mL(-1) than at lower concentrations of yeast cells. The ability of yeast cells to attach to fungal conidia or hyphae appears to be a common phenotype among phylloplane yeasts.     

Use of three screening techniques for the evaluation of fungicides to control rhizoctonia root rot of wheat

Twenty fungicides were screened for their abilities to reduce the saprophytic growth of Rhizoctonia solani on agar and in soil, and to control root rot of wheat (Triticum aestivum) in pots of soil. Thirteen showed some activity against the in vitro growth of R. solani, the most promising being Sandoz 619F and flutolanil, although none of these reduced saprophytic growth of the fungus in soil. Only benomyl and thiabendazole, which were ineffective on agar and against disease in pots, reduced saprophytic growth in soil. None of the fungicides reduced the number of plants infected in the pot experiments and only six (flutriafol, diniconazole, Schering 539865, propiconazole, Bayer HWG1608 and flutolanil) of the thirteen active on agar (+ triadimefon which was not) reduced root rot severity. The results of this study indicate the differing reactions towards R. solani of chemicals in the three screening tests and confirm the potential for chemical control of rhizoctonia root rot of wheat.     

Enhancement of Disease Control Efficacy of Chemical Fungicides Combined with Plant Resistance Inducer 2,3-Butanediol against Turfgrass Fungal Diseases

Turfgrass, the most widely grown ornamental crop, is severely affected by fungal pathogens including Sclerotinia homoeocarpa, Rhizoctonia solani, and Magnaporthe poae. At present, turfgrass fungal disease management predominantly relies on synthetic fungicide treatments. However, the extensive application of fungicides to the soil increases residual detection frequency, raising concerns for the environment and human health. The bacterial volatile compound, 2,3-butanediol (BDO), was found to induce plant resistance. In this study, we evaluated the disease control efficacy of a combination of stereoisomers of 2,3-BDO and commercial fungicides against turfgrass fungal diseases in both growth room and fields. In the growth room experiment, the combination of 0.9% 2R,3R-BDO (levo) soluble liquid (SL) formulation and 9% 2R,3S-BDO (meso) SL with half concentration of fungicides significantly increased the disease control efficacy against dollar spot and summer patch disease when compared to the half concentration of fungicide alone. In field experiments, the disease control efficiency of levo 0.9% and meso 9% SL, in combination with a fungicide, was confirmed against dollar spot and large patch disease. Additionally, the induction of defense-related genes involved in the salicylic acid and jasmonic acid/ethylene signaling pathways and reactive oxygen species detoxification-related genes under Clarireedia sp. infection was confirmed with levo 0.9% and meso 9% SL treatment in creeping bentgrass. Our findings suggest that 2,3-BDO isomer formulations can be combined with chemical fungicides as a new integrated tool to control Clarireedia sp. infection in turfgrass, thereby reducing the use of chemical fungicides.     

Combined application of Bacillus subtilis NJ-18 with fungicides for control of sharp eyespot of wheat

Antagonistic bacterium, Bacillus subtilis NJ-18, can biologically control several plant diseases. A combined strategy of wheat sharp eyespot control consisting of B. subtilis NJ-18 and fungicides was evaluated. Greenhouse and field tests were conducted to determine the effect of combining the B. subtilis NJ-18 with the fungicides flutolanil and difenoconazole for the control of wheat sharp eyespot caused by Rhizoctonia cerealis Van der Hoeven. The sensitivity of B. subtilis NJ-18 to fungicides flutolanil and difenoconazole was tested. The growth of NJ-18 was unaffected by flutolanil in a broth medium, and the survival of NJ-18 spores on wheat seed was unaffected by difenoconazole. In greenhouse experiments, disease control obtained with a combination of NJ-18 and either fungicide was better than the control obtained with the bacterium or fungicides alone, and some combinations of bacterium plus fungicide demonstrated a small synergistic effect in reducing disease. Similar results were obtained with repeated field experiments. In addition to providing a highly effective disease control, the use of combinations enabled a reduced level of fungicide application. Overall, the results suggest that the combination of B. subtilis strain NJ-18 and flutolanil or difenoconazole represents a promising new tool for the control of sharp eyespot of wheat.     

Effects of wheat foliar fungicides on the aphid endoparasitoid Aphidius rhopalosiphi DeStefani-Perez (Hym., Aphidiidae) on glass plates and on plants

Abstract: The side-effects of several fungicides used in wheat to control disease at heading growth stage were assessed on the aphid parasitoid Aphidius rhopalosiphi by tests conducted in the laboratory on glass plates and in the greenhouse on young wheat plants. Very few formulations containing only one active ingredient (carbendazim, cyproconazole or epoxyconazole) or combinations of two (carbendazim + cyproconazole, carbendazim + hexaconazole) were harmless to A. rhopalosiphi in the glass-plate tests. There was no apparent synergism between fungicides tested in combinations. The parasitoid mortalities in tests carried out on plants were less and chlorothalonil, epoxyconazole, fenpropidin, fenpropimorph, flusilazole, flutriafol, prochloraz, tebuconazole, tridemorph and a number of combinations (carbendazim + flutriafol, chlorothalonil + cyproconazole, epoxyconazole + tridemorph, chlorothalonil + hexaconazole, chlorothalonil + flutriafol, cyproconazole + prochloraz, epoxyconazole + fenpropimorph, fenpropimorph + propiconazole, propiconazole + tridemorph, triadimenol + tridemorph) were harmless or only slightly harmful to the aphid parasitoid. Several combinations (carbendazim + epoxyconazole, carbendazim + fenpropimorph, carbendazim + flusilazole, carbendazim + tebuconazole, chlorothalonil + fenpropimorph, chlorothalonil + flusilazole, fenpropimorph + fenpropidin, fenpropimorph + prochloraz, fenpropidin + propiconazole, fenpropidin + tebuco nazole, tebuconazole + triadimenol) were toxic for wasps on plants. The parasitoid mortalities were less on plants than on glass plates but the wasps spent less time on treated leaves and in some cases parasitism of aphids was reduced to a large extent. These results suggest that in addition to study of the direct effects of pesticides on beneficial insects (mortalities, reduction of fertility) their effects on the behaviour of the insects should also be studied. Products that induced a repellent effect need further testing in field or semi-field conditions. However, many fungicide combinations that have little or no effect on A. rhopalosiphi can protect wheat against a wide range of diseases and the results obtained in this study indicate that an appropriate and effective protection of wheat at earing growth stage can be achieved with products that have no effects on aphid parasitoids.     

Mechanism of action and fate of the fungicide chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) in biological systems: 2. In vitro reactions

The reaction characteristics of chlorothalonil with glyceraldehyde-3-phosphate dehydrogenase (GPDH), from yeast, (EC 1.2.1.12) were studied in vitro. Enzyme inhibition was related to the amount of [¹⁴C]chlorothalonil bound to the protein. Kinetics of enzyme inhibition was non-competitive for the substrate glyceraldehyde-3-phosphate (GAP) (K_i = 0.42 μM). Reversal of enzyme inhibition could not be demonstrated with the low molecular thiol dithiothreitol (DTT), although the thiol did protect the protein against the toxic action of the fungicide. Because 5,5' dithiobis-(2-nitrobenzoic) acid (DTNB) reduced the binding of ¹⁴C-labeled fungicide by approximately 90% it is postulated that chlorothalonil affects catalytic activity by reacting with the 4 sulfhydryl sites (cysteine-149) responsible for the binding of GAP. Certain reaction characteristics of the trichloromethyl sulfenyl fungicides with GPDH were found to be similar to those of chlorothalonil. However, chlorothalonil differed from those fungicides in that it did not react with non-thiol groups of either GPDH or -chymotrypsin (CT) and had a slower reaction rate with the GPDH. It is suggested that the differences in reaction rates of the fungicides are due to the molecular size and the chemical nature of the reactive toxiphores.     

Mycorrhizal growth in pure cultures in the presence of pesticides

The effects of pesticides on 64 ectomycorrhizal fungi of boreal forest trees were studied in vitro. The pesticides (fungicides: benomyl, chlorothalonil, copper oxychloride, maneb and propiconazole; herbicides: chlorthiamid, glyphosate, hexazinone, linuron and terbuthylazine; insecticide: cypermethrin) were selected as those commonly used in Nordic forest nurseries and afforestation sites. In general, the fungicides proved to be more toxic to ectomycorrhizal fungi than the herbicides and cypermethrin. The fungicides, chlorothalonil and propiconazole, had the clearest inhibitory effect on growth of mycorrhizal fungi. Conversely, maneb, glyphosate and terbuthylazine stimulated the growth of some mycorrhizal fungi. Leccinum versipelle and L. scabrum, Paxillus involutus and Cenococcum geophilum were the most sensitive ectomycorrhizal fungi to the various pesticides.     

The Influence of Exogenous Nutrients on the Abundance of Yeasts on the Phylloplane of Turfgrass

Four experiments were conducted to assess the effect of foliar applications of various nutrient solutions on the phylloplane yeast community of tall fescue (Festuca arundinacea Schreb.). In the first three experiments, increasing concentrations of sucrose (2–16%), yeast extract (0.5–2.5%), and sucrose plus yeast extract (2.5–18.5% total) were applied and the yeast colony forming units (cfu) enumerated 14 h later by dilution plating. Significant positive linear relationships were observed between the number of yeast cfu and applications of both yeast extract and sucrose plus yeast extract. Foliar applications of sucrose alone had no significant effect on yeast community abundance, indicating that phylloplane yeasts of turfgrass are not limited by the amount or availability of carbohydrates. In the fourth experiment, five different solutions were applied to tall fescue to investigate the response of the yeast community to organic and inorganic nitrogen sources. Tryptone or yeast extract, both with considerable amino acid composition, significantly increased the yeast population, while yeast nitrogen base (with or without amino acids) and ammonium sulfate had no affect on yeast abundance. These results suggest that organic nitrogen stimulate yeast community growth and development on the phylloplane of tall fescue, while carbohydrates, inorganic nitrogen, and non-nitrogenous nutrients have little positive effect.     

Influence of fungicides and insecticides on the entomogenous fungus Metarhizium anisopliae a pathogen of the vine weevil,Otiorhynchus sulcatus

In the laboratory, the fungicides chlorothalonil and zineb prevented germination of Metarhizium anisopliae conidia when incorporated into Sabouraud dextrose agar (SDA) at the commercial concentration (based on the manufacturers’ recommended rates for horticultural crops). Twelve other fungicides and six insecticides had no effect on spore germination when applied at the same rate. Mycelial growth of M. anisopliae on SDA plates containing the recommended rate of all the pesticides (except propamocarb) was reduced compared with SDA alone. Two fungicides, benomyl and carbendazim, totally inhibited growth at 0.1 times the recommended rate. Growth was also completely prevented by the fungicides etridiazole, triforine and zineb, and the insecticides dichlorvos and hostathion, at 10 times the recommended rate. In a glasshouse experiment, a prophylactic drench of M. anisopliae conidia reduced vine weevil (Otiorhynchus sulcatus) populations on Impatiens plants by 88%. This level of control was not significantly reduced by subsequent application (7 days after egg infestation) of any of the pesticides at the recommended concentration. Larval control in pots treated with M. anisopliae plus any one of the 12 fungicides and four insecticides examined, ranged from 82% to 98%. The insecticide diazinon applied alone reduced larval numbers by 100%. Two other insecticides, dichlorvos and cypermethrin, and the fungicide pyrazaphos, also reduced weevil populations by over 50%. These experiments demonstrate the limitations of laboratory based in vitro screening programmes for assessing the chemical compatibility of M. anisopliae.     

Non-target impacts of soybean rust fungicides on the fungal entomopathogens of soybean aphid

Soybean aphid, Aphis glycines, has caused serious economic damage to soybean across the North Central US since its introduction to North America in 2000. The management of another invasive soybean pest, Asian soybean rust, Phakopsora pachyrhizi, using foliar fungicide applications has the potential to impact soybean aphid populations by suppressing beneficial fungal entomopathogens. In 2005 and 2006, we applied recommended soybean rust fungicide treatments, consisting of strobilurin and triazole fungicides, to small soybean plots in two locations to assess if such applications might suppress aphid fungal epizootics. In Lamberton, MN, in 2005, during the epizootic, fungicide-treated plots averaged 2.0 ± 0.7% (mean ± SE) disease prevalence while untreated plots averaged 14.2 ± 5.6%. In 2007, we applied strobilurin and strobilurin-triazole mix fungicides to single-plant microplots either before or after release of Pandora neoaphidis, the most commonly observed aphid pathogen in 2005 and 2006. Treatments that contained a mixture of two active ingredients significantly lowered peak and cumulative aphid disease prevalence in both early and late reproductive stage soybeans indicating that fungicide mixtures used to manage soybean rust can negatively impact an aphid-specific fungal pathogen. However, no consistent soybean aphid population response was observed in these studies of low levels of aphid fungal infection.     

Control of smoulder (Botrytis narcissicold) in narcissus with fungicides

Smoulder, caused by Botrytis narcissicola, is the most widespread foliar disease of narcissus (Narcissus cultivars) in the UK and causes significant losses in bulb and flower yield. A range of current and novel fungicide treatments was examined to determine if control of the disease could be improved and to measure the effect of treatments on bulb yield. In assays on pot-grown plants, carbendazim+flusilazole, epoxiconazole, iprodione + thiophanate-methyl, tebuconazole and vinclozolin reduced lesion size by more than 90%, compared with untreated plants, when applied 1 day before inoculation with the fungus; vinclozolin and tebuconazole were also effective when applied 2 days after inoculation. In field experiments in Cambridgeshire and Lincolnshire, significant reductions were observed in secondary smoulder symptoms (leaf lesions and stem rot) following fungicide sprays. Spray programmes of the novel fungicides azoxystrobin, cyprodinil, kresoxim-methyl, tebuconazole and pyrimethanil were as effective, or more so, than the standard treatments (chlorothalonil, vinclozolin) used by growers. Mixtures of carbendazim + tebuconazole and azoxystrobin + tebuconazole also gave effective control of smoulder. Programmes of four to six sprays, using two or three fungicides with different modes of action, applied alternately, reduced smoulder by 35–69% and increased bulb yields by 7–59%. Although treatment around and after flowering resulted in the greatest control of secondary smoulder, treatment before flowering was required for the highest bulb yields. The choice of fungicides for use in spray programmes to provide effective and reliable control of smoulder, and the possibility of control using fewer sprays, are discussed.     

Assessment of phylloplane yeasts on selected Mediterranean plants by FISH with group- and species-specific oligonucleotide probes

A previous culture-dependent survey of phylloplane yeasts from selected Mediterranean plants showed that a few species were present in high densities in almost all leaf samples, regardless of the plant type, location or sampling season. However, a few species appeared to be restricted to Cistus albidus leaves, namely Cryptococcus cistialbidi . Here, we describe a culture-independent FISH assay to detect and quantify whole yeast cells in leaf washings. After optimization, the technique was used to check the apparent association between C. albidus leaves and C. cistialbidi and the abundance and ubiquity of other basidiomycetous yeast species such as Erythrobasidium hasegawianum and Sporobolomyces spp. in leaf samples from this and other neighboring plants ( Acer monspessulanum and Quercus faginea ). No yeast cells were detected in Pistacia lentiscus leaf samples. We were also able to demonstrate that three phylloplane yeasts ( C. cistialbidi, E. hasegawianum and Sporobolomyces spp.) appeared to be log-normally distributed among individual C. albidus leaves. The log-normal distribution has important implications for the quantification of phylloplane yeasts based on the washing and plating of bulk leaf samples, which will tend to overestimate the size of the respective populations and become an error source in yeast surveys or related biocontrol studies.     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Impact of fungicides on Metarhizium anisopliae in the rhizosphere, bulk soil and in vitro

The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) is registered in the United States and The Netherlands for black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) control in container-grown ornamentals. These studies were conducted to determine the compatibility of M. anisopliae (F52) with a wide range of fungicides commonly applied to container-grown ornamentals for the management of soil-borne plant pathogens. The impact of fungicides on spore germination and mycelial growth were determined in vitro. In addition, M. anisopliae persistence in bulk and rhizosphere soil was determined 30 days following dual application of each fungicide at 7–28 days intervals as prescribed. A number of fungicides (thiophanate-methyl, dimethomorph, captan, triflumizole, triflozystrobin, pyraclostrobin, azoxystrobin) inhibited spore germination in vitro. A larger number of fungicides (fosetyl-AI, thiophanate-methyl, dimethomorph, captan, quintozene, triflumizole, fludioxanil, triflozystrobin, pyraclostrobin, fludiox-mefanox, iprodione, azoxystrobin, phosphorus acid/K-salts) inhibited mycelial growth in vitro. Only three fungicides (etridiazole, propamocard and mafanoxam) had no significant impact in vitro on spore germination or mycelial growth. While a number of fungicides had a detrimental impact in vitro, there was no impact on M. anisopliae populations in bulk soil following dual application of any fungicide. However, the fungicides captan and triflumizolet, which have a short reapplication interval, had a detrimental impact on M. anisopliae populations in the rhizosphere. As researchers develop rhizosphere competence as an alternative management strategy for black vine weevil, the fungicides captan and triflumizole should be avoided.