Inoculum prevalence, host infection and biological control of Colletotrichum acutatum: causal agent of blueberry anthracnose in British Columbia

To identify the causal organism of anthracnose (ripe-rot), which reduces yield and postharvest quality of blueberries grown in British Columbia, Canada, 80 isolates were recovered from diseased fruits collected from commercial blueberry fields during 2002–04 and identified as Colletotrichum acutatum using colony morphology, growth rate and species-specific PCR primers. In vitro incubation of replicated sets of inoculated detached berries at various temperatures produced infection at temperatures of 7–30°C, with an optimum at 20°C. Colletotrichum acutatum could not survive on the soil surface as mummified berries but the pathogen was detected mostly within flower buds and less so in blueberry twigs and fruit trusses. Infection of developing flower buds in May–June of the preceding growing season gave the highest inoculum recovery in the following year. Two commercial fungal biocontrol agents, Prestop ( Gliocladium catenulatum ) and PlantShield ( Trichoderma harzianum ), each reduced anthracnose development in 2003 and 2004 by up to 45% when sprayed three times onto plants between flowering and fruit ripening.     

Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea

ABSTRACT Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic low-molecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases.     

Evaluation of Microbial Antagonists for Biological Control of Botrytis cinerea Stem Infection in Cucumber and Tomato

Of fifteen isolates of yeasts, filamentous fungi and bacteria and a commercial product, tested in a bioassay with stem segments, eleven isolates consistently reduced incidence of disease and sporulation of Botrytis cinerea Pers; Fr in tomato and seven isolates in cucumber. Several isolates reduced disease by more than 75% in all experiments. Six antagonists that performed well in the bioassays and that were fairly easy to produce in vitro, were selected for further testing in two glasshouse experiments with cucumbers. After application of spores of B. cinerea and the antagonists or the fungicide tolylfluanid to pruning wounds, disease incidence was reduced by 50–100% by all antagonists in both experiments and only in one experiment by tolylfluanid.For Trichoderma harzianum T39, Aureobasidium pullulans and Cryptococcus albidus, biological control efficacy in bioassays with cucumber stem segments was not strongly influenced by temperatures in the range between 18 and 30°C, but at 24°C the efficacy of the three antagonists strongly decreased at relative humidities of 90% and 80% (vapour pressure deficits 0.299 and 0.598kPa, respectively) compared to 100.     

Control of Glomerella cingulata f.sp. camelliae with fungicides

A critical test of fungicidal activity against the wound pathogen Glomerella cingulata f.sp. camelliae on camellias involved the application of a conidial suspension to scratches on detached leaves, treated with fungicide 24 h previously. In this test, prochloraz at 250–500 μg/ml a.i. in both EC and manganese WP formulations was highly effective, inhibiting leaf rotting by 90–100% compared with 55% or less in some tests with benomyl, chlorothalonil and captafol at 500–1000 μg/ml and captan at 1200 μg/ml. Prochloraz gave similar protection to leaves when applied to whole plants. Protection was less, especially on older leaves, when wounding and inoculating were delayed by 7 or more days after treatment. Prochloraz did not prevent all infection through comparatively large wounds, e.g. leaf scars, made on stems 24 h after treatment.
Poor control of the disease in a nursery was not due to resistance of the pathogen to benomyl; in vitro tests failed to detect full resistance at this time. Under nursery conditions a statutory policy of destroying infected plants and spraying the remainder with prochloraz or prochloraz manganese (both at 500 μg/ml), alternating with benomyl (500 μg/ml) on a weekly basis apparently prevented spread of the disease to healthy plants.     

Screening for potential antagonists of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Bacteria isolated from wheat seedlings, plants or straw from several field sites were screened for antagonism towards the cereal eyespot pathogen Pseudocercosporella herpotrichoides on several media of differing nutrient status. Thirteen out of 348 isolates inhibited pathogen growth on low-nutrient media and several also prevented spore germination or reduced germ tube extension. These were selected for further tests on wheat seedlings inoculated with the eyespot fungus. Twelve known bacterial antagonists of other fungal plant pathogens were tested in vitro using the same methods, and the majority showed some activity towards P. herpotrichoides. Selected isolates were equally inhibitory to both W and R pathotypes of the fungus. Effects of potential antagonists on disease development were assessed by scoring lesions or by counting the number of infection plaques formed by the fungus on leaf sheaths. Two isolates of Pseudomonas fluorescens , along with a commercial strain of Streptomyces griseoviridis , showed activity both in vitro and in subsequent infection trials with plants and may therefore be of potential value as antagonists of P. herpotrichoides.     

Biological control ofBotrytis cinerea on tomato stem wounds withTrichoderma harzianum

The effectiveness ofTrichoderma harzianum in suppression of tomato stem rot caused byBotrytis cinerea was examined on tomato stem pieces and on whole plants. Ten days after simultanous inoculation withB. cinerea andT. harzianum, the incidence of infected stem pieces was reduced by 62–84%, the severity of infection by 68–71% and the intensity of sporulation by 87%. Seventeen days after inoculation of wounds on whole plants, the incidence of stem rot was reduced by 50 and 33% at 15 and 26 °C, respectively, and the incidence of rot at leaf scar sites on the main stem was reduced by 60 and 50%, respectively. Simultanous inoculation and pre-inoculation withT. harzianum gave good control ofB. cinerea (50 and 90% disease reduction, 10 days after inoculation). The rate of rotting was not reduced by the biocontrol agent once infection was established. However, sporulation byB. cinerea was specifically reduced on these rotting stem pieces. Temperature had a greater effect than vapour pressure deficit (VPD) on the efficacy of biocontrol. Suppression ofB. cinerea incidence byT. harzianum on stem pieces was significant at 10 °C and higher temperatures up to 26 °C. Control of infection was significantly lower at a VPD of 1.3 kPa (60% reduction), than at VPD<1.06 kPa (90–100% control). Reductions in the severity of stem rotting and the sporulation intensity of grey mould were generally not affected by VPD in the range 0.59–1.06 kPa. Survival ofT. harzianum on stems was affected by both temperature and VPD and was greatest at 10 °C at a low VPD and at 26 ° C at a high VPD.     

Fusarium crown and root rot of tomatoes in the UK

Fusarium crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici was found in the UK in 1988 and 1989 mainly in rockwool-grown tomato crops. Up to 14% of plants were affected in individual crops. In experiments, leaf and stem symptoms did not appear until the time of first fruit harvest even when the plants were inoculated at planting, first flowers or fruit set. Conidial inoculum at 10⁶ spores/plant applied at seed sowing killed 70–83% of tomato seedlings, whereas similar levels of inoculum applied to young plants caused root and basal stem decay, and eventually death but only after fruit harvest began. Disease incidence and symptom severity increased with inoculum concentration. Experimentally, the disease was more severe in peat- or compost-grown plants than in rockwool. Disease spread was only a few centimetres in 50 days in experimental rockwool-grown plants. All tomato cultivars tested were highly susceptible. Prochloraz-Mn was highly effective against the pathogen in vitro and controlled the disease in the glasshouse, but only when applied preventively. Non-pathogenic Fusarium oxysporum isolates and Trichoderma harzianum also reduced FCRR disease levels.     

健康与感染青枯病烟株根际土壤与茎秆真菌群落结构与多样性

为研究感染青枯病后烟株根际土壤与茎秆真菌群落结构与多样性的变化,对健康和感染青枯病烟株的根际土壤、病株茎秆发病组织和健株茎秆健康组织等样品中真菌ITS区的rDNA进行了PCR扩增、用Illumina MiSeq测序技术对扩增DNA片段进行高通量测序,并分析不同样品的真菌群落组成与多样性。结果表明,所有烟株根际土壤中优势门为子囊菌门Ascomycota和接合菌门Zygomycota;所有茎秆样品中优势门为担子菌门Basidiomycota和子囊菌门。在属水平,被孢霉属Mortierella、镰刀菌属Fusarium和隐球菌属Cryptococcus为所有土壤中的主要菌属,Boeremia主要存在于发病烟株根际土壤中,而木霉属Trichoderma主要存在于健康烟株根际土壤。发病茎秆病害组织中优势属为小画线壳属Monographella、隐球菌属、鬼伞属Coprinopsis和赤霉属Gibberella;发病茎秆病健交界处组织中优势属为隐球菌属、红酵母属Rhodotorula和小画线壳属。健康烟株茎秆组织中优势属为隐球菌属、链格孢属Alternaria和红酵母属;健康烟株中与发病茎秆病健交界处组织等高茎秆中优势属为镰刀菌属、隐球菌属、链格孢属和Gibellulopsis。青枯菌侵染烟株后根际土壤、发病茎秆病害组织和发病茎秆病健交界处组织的真菌群落中物种丰富度与多样性均显著提高,且发病茎秆病害组织与发病茎秆病健交界处组织真菌群落的变化大于根际土壤。研究结果为烟草青枯病的生物防治提供了参考。     

Pectolytic and Cellulolytic Enzymes of Dicarboximide-sensitive and Resistant Strains of Botrytis cinerea1

Starting in 1979 the pathogenicity of dicarboximide-resistant and sensitive strains of Botrytis cinerea was regularly examined in greenhouse tests using grape plants. In these tests the proportion of resistant strains with low or nearly no pathogenicity was always higher than that of sensitive strains. This led to comparative studies on the enzyme activities of sensitive and resistant field isolates. Pectolytic and cellulolytic enzymes were chosen because of their importance in the infection process of B. cinerea. The results of these studies indicated that no correlation could be found between the activity of these enzymes and the pathogenicity of the tested isolates, and that resistant strains tended to have higher enzyme activities than sensitive ones. Comparison of the enzyme activities of laboratory-adapted isolates and the original sensitive ones gave similar results. Since enzyme activities do not seem to play an important role in explaining the general observations on the slow increase and spread of resistant strains in vineyards, other factors must be considered, such as the stability of the dicarboximide resistance, which apparently is very low.     

Evaluation of fungal antagonists for control of onion white rot in soil box trials

Four fungi ( Chaetomium globosum, Trichoderma viride, T. harzianum, Trichoderma sp.) were capable of reducing the incidence of onion white rot relative to the untreated control in two soil-box trials. When applied as a soil additive (sand: bran: fungal homogenate, 1:1:2) at the rate of 0-1% wheat bran/g dry soil, all fungal isolates provided levels of disease control equivalent to the fungicide (procymidone 0-5 g a.i./100g seed) treatment. The best results were achieved with the Chaetomium globosum and Trichoderma (C62) isolates which gave 78% and 73% control of white rot. respectively, in trial 1 and 67% and 73% control, respectively, in trial 2. Reduced control was observed when the test fungi were applied as seed coatings or incorporated into alginate pellets.     

Persistent,symptomless, systemic,and seed-borne infection of lettuce by <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Experiments are presented which show that Botrytis cinerea, the cause of grey mould disease, is often present in symptomless lettuce plants as a systemic, endophytic, infection which may arise from seed. The fungus was isolated on selective media from surface-sterilised sections of roots, stem pieces and leaf discs from symptomless plants grown in a conventional glasshouse and in a spore-free air-flow provided by an isolation propagator. The presence of B. cinerea was confirmed by immuno-labelling the tissues with the Botrytis-specific monoclonal antibody BC-12.CA4. As plants grew, infection spread from the roots to stems and leaves. Surface-sterilisation of seeds reduced the number of infected symptomless plants. Artificial infection of seedlings with dry conidia increased the rate of infection in some experiments. Selected isolates were genetically finger-printed using microsatellite loci. This confirmed systemic spread of the inoculating isolates but showed that other isolates were also present and that single plants hosted multiple isolates. This shows that B. cinerea commonly grows in lettuce plants as an endophyte, as has already been shown for Primula. If true for other hosts, the endophytic phase may be as important a component of the species population as the aggressive necrotrophic phase.     

The influence of inoculum concentration, relative humidity, and temperature on infection of greenhouse tomatoes by Botrytis cinerea

Botrytis cinerea causes serious crop losses in greenhouse tomato crops through infection of flowers and stem wounds. Experiments were carried out to determine the effects of inoculum concentration, relative humidity (RH), and temperature at these two infection sites. Infection of permanent flower parts increased as a function of inoculum concentration and both length of exposure to high RH (approximately 100% for 0–36 h) and specified continuous RH (56–100%). A low level of infection was still evident under continuous 56% RH. Interruption of periods of high RH with breaks of low RH did not reduce infection. Infection of stem wounds was less dependent on inoculum concentration or RH. Factorial combinations of inoculum concentration, RH, and temperature produced significant interactions. Higher temperature increased infection of flowers but reduced infection of stem wounds. The main implications for control in commercial crops are as follows. Lowering the aerial spore concentration by maintaining the disease at a low level will reduce flower infection. Lowering RH will reduce but not eliminate flower infection but will have only a small effect on stem infection. Raising the temperature (from 15 to 25°C) will reduce stem infection, and whilst flower infection increases, this is counteracted by increased flower production and a decrease in the proportion of infections reaching the peduncle and stem.     

Application of alginate gel for protection of wounds against crown gall (Agrobacterium tumefaciens)

In glasshouse and laboratory experiments, calcium alginate gel, produced in situ on wounds by successive dipping in solutions of sodium alginate and CaCl₂ gave significant protection against infection by pathogenic isolates of Agrobacterium tumefaciens on carrot discs, tomato cuttings and chrysanthemum cuttings. Gel protection was sometimes equal to that given by strain K84 of A. radiobacter and also operated against a pathogenic isolate insensitive to control by strain K84. Sodium alginate or CaCl₂ alone gave little or no protection, suggesting that calcium alginate acted by physically excluding the pathogen from wound surfaces. The use of the method described is discussed for delivery of biological or chemical control agents to plant surfaces whilst also providing protection in its own right.     

Control of infection and sporulation ofBotrytis cinerea on bean and tomato by saprophytic bacteria and fungi

Sixty isolates of saprophytic microorganisms were screened for their ability to reduce the severity of grey mould (Botrytis cinerea) infection and sporulation. Isolates of the bacteriaXanthomonas maltophilia, Bacillus pumilus, Lactobacillus sp., andPseudomonas sp. and the fungusGliocladium catenulatum reduced germination of conidia of the pathogen and controlled disease on bean and tomato plants. Their activity under growth room conditions was good, consistent, and similar to the activity of the known biocontrol agent,Trichoderma harzianum T39 (non-formulated). Although the tested isolates may for nutrients with the germinating conidia ofB. cinerea, resistance induced in the host by live or dead cells were also found to be involved. Inhibitory compounds were not detected on treated leaves. Sporulation ofB. cinerea after its establishment on leaves was also reduced by the above mentioned isolates and byPenicillium sp.,Arthrinium montagnei, Ar. phaeospermum, Sesquicillium candelabrum, Chaetomium globosum, Alternaria alternata, Ulocladium atrum, andT. viride. These sporulation-inhibiting fungi did not reduce the infection of leaves byB. cinerea. Most of these selected fungi and bacteria were capable of reducing lesion expansion.     

Biological control of fire blight of hawthorn (Crataegus monogyna) with Erwinia herbicola under protected conditions

Isolates of Erwinia herbicola , obtained from flowers and leaves of hawthorn ( Crataegus monogyna) , were screened as potential control agents of fire blight disease (caused by Erwinia amylovora) using an immature pear fruit assay. Selected isolates were subsequently tested for disease control by infection of hawthorn blossom in the laboratory, and by shoot infection of hawthorn plants grown under controlled (glasshouse) and fluctuating (polythene tunnel) environmental conditions.
Although the immature pear fruit assay provided a general screen for the selection of antagonists for the control of both blossom and shoot blight, it had two major limitations when quantitatively applied. Firstly there were inconsistencies in the relative effects of different isolates on the pear-slice surface, with some isolates being more suppressive than the standard antagonist Eh252 in the first screening and less in the second. Secondly the assay was not able to predict accurately the level of control in the intact plant-as no correlation occurred between the level of control in the pear fruit assay and the percentage control of either blossom blight or shoot blight.
Two isolates of E. herbicola , WL9 and WL40, reduced both blossom- and shoot-blight. WL9 provided over 80% control of blossom blight, equivalent to that provided by chemical agents, and also gave total control of shoot blight when applied at a WL9: pathogen ratio of 10:1.     

The use of Trichoderma species to control strawberry fruit rots

The effect of temperature on the growth and antagonistic properties of Trichoderma species against Botrytis cinerea and Mucor mucedo (strawberry fruit pathogens) was studied. Five strongly antagonistic isolates were further used in field experiments. The incidence of pre-harvest rots caused by B. cinerea and the rate of post-harvest spoilage were similarly reduced when strawberry flowers were sprayed either with the fungicide dichlofluanid or with spores of selected Trichoderma isolates.     

Occurrence of fungal pathogens of carrots on wooden boxes used for storage

Infested wooden boxes, previously used for carrot storage, were sampled in four commercial carrot production farms in Bradford Marsh, Ontario, and screened for fungal occurrence. At least 128 and 465 fungal isolates were recovered from these boxes in 2001 and 2002, respectively, and were classified into 10 taxonomic groups, including Alternaria spp., Aspergillus spp., Botrytis cinerea , Fusarium spp., Mucor spp., Penicillium spp., Rhizoctonia carotae , Rhizopus spp., Sclerotinia sclerotiorum and Trichoderma spp. A subsample of 27 putative pathogenic isolates was further tested for the ability to cause disease on carrots and to colonize wood surfaces under growth room and cold storage conditions. Approximately 60% of the taxa growing on wood caused lesions upon contact with intact carrots in cold storage. Isolates of S. sclerotiorum , B. cinerea and R. carotae caused the most severe diseases, developed most extensively on wooden surfaces in cold storage, and represented 12% of the recovered fungi. Isolates of Alternaria spp., Aspergillus spp., Fusarium spp., Mucor spp., Penicillium spp., Rhizopus spp. and Trichoderma spp. caused negligible or no disease on carrots and represented 88% of recovered fungi. Several of these fungi, however, showed potential to colonize wooden surfaces and cause disease on sliced carrots. This study suggests that pathogenic inocula occurring on used wooden boxes can initiate disease upon contact with healthy carrots and reusing infested boxes can affect carrots in storage.     

Isolation and antifungal activity of kakuol, a propiophenone derivative from Asarum sieboldii rhizome

An antifungal substance active against Colletotrichum orbiculare (Berk & Mont) Arx was isolated from the methanol extracts of Asarum sieboldii (Miq) Maek rhizomes. High-resolution MS, NMR and UV spectral data confirmed that the antifungal substance is kakuol, 2-hydroxy-4,5-methylenedioxypropiophenone. Colletotrichum orbiculare was most sensitive to kakuol, with MIC of 10 microg ml(-1). Kakuol also completely inhibited the mycelial growth of Botrytis cinerea Pers ex Fr and Cladosporium cucumerinum Ellis & Arthur at 50 microg ml(-1) and 30 microg ml(-1), respectively. However, no antimicrobial activity was found against yeast and bacteria even at 100 microg ml(-1). Kakuol exhibited a protective activity against the development of anthracnose disease on cucumber plants. The control efficacy of kakuol against the anthracnose disease was in general somewhat less than that of the commercial fungicide chlorothalonil. This is the first report to demonstrate in vitro and in vivo antifungal activity of kakuol against C. orbiculare infection.     

Evaluation of potential biocontrol agents against Claviceps africana in vitro and in vivo

Undiluted culture filtrates of two commercial products of Trichoderma spp., Trichopel and Trichoflow, and two isolates of Penicillium citrinum completely inhibited the conidial germination of macroconidia of Claviceps africana , the cause of ergot or sugary disease of sorghum ( Sorghum bicolor ) in vitro . Similarly, Pseudomonas aeruginosa and Burkholderia cepacia completely inhibited macroconidial germination, with the former being more effective at high dilutions. In contrast, these bacterial isolates failed to inhibit infection in vivo in glasshouse tests with ergot-inoculated sorghum, but all fungal biocontrol agents (including an isolate of Epicoccum nigrum ) reduced the severity of disease (percentage of infected spikelets per panicle), in some cases completely inhibiting the development of ergot. In a second glasshouse trial, optimum control was achieved when the biocontrol agents were applied 3–7 days before inoculation with conidia of C. africana .     

Histopathological aspects of mango resistance to the infection process of Ceratocystis fimbriata

Mango wilt, caused by Ceratocystis fimbriata, is one of the most important diseases affecting mango yields in Brazil. Information regarding the infection process of C. fimbriata in the stem tissues of mango from different cultivars and the basis of host resistance to the pathogen is rare in the literature. Thus, the objective of the study was to investigate how infection by two isolates of C. fimbriata can be affected by mango cultivar‐specific mechanisms of resistance. Disease progress on the inoculated stem tissues of the mango cultivars was evaluated and stem sections were obtained from the site of inoculation and prepared for histopathological observations using light microscopy. The factors mango cultivars and C. fimbriata isolates and their interaction were significant for all measures of disease development. Plants from the cultivars Espada, Haden and Palmer inoculated with isolates of C. fimbriata were more susceptible, whereas plants from the cultivars Tommy and Ubá were moderately resistant and resistant, respectively. Histopathologically, fungal isolates apparently massively colonized the stem tissues of plants from the susceptible cultivars Espada, Haden and Palmer, starting from the collenchyma and moving in the direction of the cortical parenchyma, xylem vessels and pith parenchyma. By contrast, on stem tissues of plants from the resistant cultivars Tommy Atkins and Ubá, most of the cells reacted to C. fimbriata infection by accumulating amorphous material. The results from the present study strongly indicated the importance of phenolic compounds for mango cultivar resistance against infection by Brazilian C. fimbriata isolates.