Correlative analysis of Mycosphaerella graminicola pathogenicity and cell wall-degrading enzymes produced in vitro: the importance of xylanase and polygalacturonase

Eight Mycosphaerella graminicola isolates were investigated for correlations between pathogenicity and the in vitro production of cell wall-degrading enzymes. Isolate pathogenicity was evaluated in terms of lesion and production of pycnidia in wheat leaves. Additionally, the isolates were compared over time for their ability to produce in vitro significant levels of xylanase (EC 3·2·1·8), β-xylosidase (EC 3·2·1·37), β-1,3-glucanase (EC 3·2·1·6), cellulose (EC 3·2·1·4) and polygalacturonase (EC 3·2·1·15) activities when grown in a liquid medium. Correlation tests and principal component analysis revealed a significant correlation between the in vitro production of xylanase and pectinase and pathogenicity components. Xylanase was correlated to necrosis frequency ( r  = 0·795), β-xylosidase was correlated to the mean of the lesion length ( r  = −0·787), whereas polygalacturonase was correlated to the time when 50% of the leaves contained a lesion ( r  = 0·776), the lesion frequency ( r  = 0·646) and the time when 50% of the leaves showed pycnidia ( r  = −0·711). The results suggest that these two groups of cell wall-degrading enzymes are therefore likely to be key determinants of pathogenicity in M. graminicola .     

Trichothecenes and aggressiveness of Fusarium graminearum causing seedling blight and root rot in cereals

Greenhouse trials conducted in 2003 and 2004 investigated the impact of trichothecenes on the severity of seedling blight and root rot in common wheat ( Triticum aestivum ), durum wheat ( Triticum turgidum var. durum ), barley ( Hordeum vulgare ) and triticale (× Triticosecale 6x ) using two trichothecene-producing and two trichothecene-nonproducing Fusarium graminearum strains. In 2003 seedling emergence and survival following soil infestation of the trichothecene-producing strain (Gz3639) were significantly reduced compared with the trichothecene-nonproducing strain (GzT40), while root-rot incidence and severity were increased significantly. In 2004, two trichothecene-producing strains (Gz3639 and GzT106) reduced seedling emergence and survival ( P  ≤ 0·01) in eight of 10 crops/cultivars based on single-degree-of-freedom contrasts. However, when results from all strains were combined no significant differences were observed between two trichothecene-producing and two trichothecene-nonproducing F. graminearum strains. Inoculation with GzT106, a trichothecene-producing 'add-back' strain, resulted in more severe root rot symptoms in eight of 10 cultivars ( P  ≤ 0·01–0·05) and lower seedling emergence and survival in seven of 10 cultivars ( P  ≤ 0·01–0·10), compared with the wild-type parental strain Gz3639. The presence of trichothecenes may play an important role in the aggressiveness of F. graminearum .     

Sulcotrione soil persistence and mobility in summer maize and winter wheat crops

Sulcotrione soil persistence in spring maize ( Zea mays L.) crops grown on a sandy loam soil was greater at pH 5·5 and 6·0 (soil half-life T _1/2≈58 days) than at pH 7·1 ( T _1/2 = 44 days). Sulcotrione was also applied as recommended on a summer maize crop at the five- to six-leaf growth stage, grown on a sandy loam soil. Sulcotrione soil half-life was 44 days, and the herbicide remained mainly in the 0- to 5-cm surface soil layer during the cropping period, in spite of the high water solubility and the heavy rains at the end of August; lower sulcotrione concentrations (10–18% of the total during the 2-month period after sulcotrione application) were detected in the 5- to 10-cm surface soil layer. The herbicide was applied pre-emergence to winter wheat ( Triticum aestivum L.) at four sites that differed in their soil texture and composition: loamy sand, sandy loam, loam and clay loam. Persistence was greater in the soils containing more organic matter. In soils having similar organic matter contents, persistence was lower in the soil containing more sand relative to loam and clay. During the winter crops, sulcotrione moved down to the 10- to 15-cm soil layer, in spite of the fact that the rains were lower in winter than in summer. Sulcotrione most generally was not detected in the 15–20 cm soil layer of the maize and winter wheat crops.     

Effect of temperature on head blight of wheat caused by Fusarium culmorum and F. graminearum

The effect of small temperature differentials (16 vs. 20°C) on the pathogenicity of deoxynivalenol producing single isolates of Fusarium culmorum and F. graminearum and on the fusarium head blight (FHB) response of eight wheat cultivars was examined. Fusarium culmorum inoculation caused greater visual disease symptoms at 20°C than at 16°C, both overall and on an individual cultivar basis (overall AUDPC = 13·5 and 9·6, respectively) ( P  < 0·05). In contrast, F. graminearum inoculation caused greater overall visual disease symptoms at 16°C than at 20°C, both overall and at the individual cultivar level (overall AUDPC = 12·8 and 10·9, respectively) ( P  < 0·05). Results showed both F. culmorum and F. graminearum inoculations caused a greater loss in yield at 20°C (54·3 and 46·9% relative 1000-grain weight, respectively) compared with 16°C (73·3 and 66·9% relative 1000-grain weight, respectively) ( P  < 0·05). Fusarium culmorum -inoculated heads contained similar amounts of fungal DNA at both 16 and 20°C (1·9 and 1·7 ng mg⁻¹ of plant material, respectively) (not significant), while for F. graminearum inoculation, plants contained higher amounts of fungal DNA at 20°C (2·0 and 1·0 ng mg⁻¹ of plant material, respectively) ( P  < 0·05). Overall, there was a significant negative correlation between AUDPC and percentage relative 1000-grain weight at both 16 and 20°C ( r  =−0·693 and −0·794, respectively, P  < 0·01).     

Infection and colonization of melon roots by Monosporascus cannonballus in two cropping seasons in Arizona and California

Although canopy collapse of melons (one of the above-ground symptoms of vine decline caused by Monosporascus cannonballus ) occurred late in the growing season, the onset of root infection occurred much earlier. In three early winter-spring and two late winter-spring crops, the onset of root infection occurred 47–65 and 35–36 days after planting, respectively. In contrast, in four summer-autumn crops, the onset of root infection occurred within 9–17 days after planting. Vine decline occurred commonly in winter-spring crops, but did not occur in any of the summer-autumn crops. Following the onset of root infection, the percentage of plants infected increased at rates of 0·031–0·036 and 0·038–0·070 per unit per day for winter-spring and summer-autumn crops, respectively, based on the monomolecular disease progress model. Root lesions were first observed 14–42 days after the onset of infection in winter-spring crops, and 14–28 days after the onset of infection in summer-autumn crops. Pathogen reproduction occurred primarily at the end of each growing season.     

The occurrence in Denmark of black root rot of pea caused by Thielaviopsis basicola

Thielaviopsis basicola has been shown to be a root pathogen of pea of considerable importance in Denmark. The fungus is only found in fields with one or more previous pea crops in the field history. In the dry and warm growing season of 1989 the fungus was found in 0·6% and 3·2% of the fields in two separate areas in Denmark. In the fields where T. basicola was detected the average disease severity index in plant samples was 51·8, whereas the average disease severity index in plant samples without the fungus was 27·0. The average yield of green peas was reduced from 5167 to 4171 kg/ha when T. basicola was present. For detection and isolation of T. basicola it is important to use a technique combining microscopic examination, a semi-selective medium and a dilution plate method.     

Foliar resistance of cacao (Theobroma cacao) to Phytophthora palmivora as an indicator of pod resistance in the field: the effect of light intensity and time of day of leaf collection

Resistance of cacao leaves to Phytophthora palmivora was studied with regard to the time of leaf collection (morning, afternoon) and the degree of exposure of the leaves to light in the field (low, medium and high). The efficiency of leaf disc inoculations in predicting field resistance of nine clones was compared with that of detached and attached pod inoculations. Significant effects were observed, with leaves exposed to high light intensity and collected early in the afternoon showing highest susceptibility. The effect of time of leaf collection was reduced when leaves were stored overnight and leaf discs prepared and inoculated the following day, as compared to inoculations on the day of collection. Interactions between the main factors were significant, though less substantial than the clone effects. The most significant correlations with pod resistance ( r  = 0·70 to 0·97) were obtained for leaves collected early in the morning and exposed to intermediate shade conditions in the canopy. For other treatments, the correlations with pod resistance were still positive ( r  = 0·23 to 0·83) but often not significant. Pod inoculations in the laboratory were better correlated with field resistance ( r  = 0·92) than pod inoculations in the field ( r  = 0·72). Detached pod inoculations were also better correlated with leaf disc inoculations than those of attached pods. The results confirm the validity of laboratory inoculations of leaves and pods to assess field resistance to Phytophthora . Standardization of the leaf disc test is essential to obtain reliable results.     

Effects of nitrogen, phosphorus, potassium and calcium nutrition on strawberry anthracnose

The effects of a range of concentrations of four nutrients – nitrogen, phosphorus, potassium and calcium – in fertilizer solutions on the severity of anthracnose on strawberry cv. Nyoho cultivated under a noncirculation hydroponics system were determined after inoculation with Colletotrichum gloeosporioides . Crop growth and tissue nitrogen, phosphorus, potassium and calcium contents of the entire above-ground parts of the plant were also investigated. Elevated nitrogen and potassium concentrations in the fertilizer solution increased disease severity in contrast to phosphorus and calcium. Treatment with either NH₄ or NO₃ nitrogen was not significantly different. The dry weight of the strawberry plants increased significantly with elevated concentrations of nitrogen ( R ² = 0·9078) and phosphorus ( R ² = 0·8842), but was not influenced by the elevated amounts of potassium ( R ² = 0·8587) and calcium ( R ² = 0·6526) concentrations.     

Effect of temperature on latent period of septoria leaf blotch on winter wheat under outdoor conditions

Batches of two winter wheat cultivars (Riband and Apollo) were inoculated with conidia of Mycosphaerella graminicola at weekly intervals over a 2 year period. Following 72 h incubation, plants were placed in ambient temperatures ranging between −7 and 32°C with mean batch temperatures of 2·9–20·2°C. Latent period until the first visible symptoms ranged between 11 and 42 days. The relationship between development of lesions and accumulated thermal time was described using a shifted cumulative gamma distribution model. The model provided good estimates of lesion development with r ² > 0·92 for both cultivars. Base temperatures, below which the pathogen did not develop, were estimated from the model as approximately −2·4°C for the two cultivars. Latent period was estimated as being 250 and 301 degree-days above the estimated base temperature, when defined as time from inoculation to first lesion and time to 50% of maximal lesions, respectively, for cv. Riband. The values for cv. Apollo were similar, but with estimates of thermal time periods c . 5% higher. The relationship between mean temperature and inverse latent period, expressed as days either to first lesion or to 50% of maximal lesions, was best described by a linear regression with r ² > 0·96 for both cultivars. The opportunity for plants to outgrow disease was reduced when prolonged periods of cold temperature occurred, because the base temperature for growth of the pathogen was less than that for the crop.     

Genetic variation and covariation for aggressiveness, deoxynivalenol production and fungal colonization among progeny of Gibberella zeae in wheat

Gibberella zeae (anamorph Fusarium graminearum ) causes head blight of cereals and contaminates grains with mycotoxins such as deoxynivalenol (DON). To determine the correlations among aggressiveness traits, fungal colonization and DON production, 50 progeny from a segregating population of G. zeae were inoculated onto a susceptible winter wheat cultivar in three field environments (year–location combinations). Aggressiveness traits were measured as head-blight rating and plot yield relative to noninoculated plots. Fungal colonization, measured as Fusarium exoantigen (ExAg) content, and DON production were analysed with two ELISA formats. Disease severity was moderate to high based on head-blight rating and relative plot yield. Fusarium ExAg content and DON production ranged from 0·26–1·41 units and from 4·18–43·70 mg kg⁻¹, respectively. Significant ( P  = 0·01) genotypic variation was found for all traits. Heritability for Fusarium ExAg content was rather low because of high progeny–environment interaction and error. DON/ Fusarium ExAg ratio did not vary significantly ( P  > 0·1) among progeny. Correlation between DON production and Fusarium ExAg content across environments was high ( r  = 0·8, P  = 0·01), but no covariation existed between aggressiveness traits and DON/ Fusarium ExAg content ratio.     

Interactions of asparagns root tissue with soil microorganisms as a factor in early decline of asparagus

Sterilized root residues of asparagus added at a rate of up to 20gkg^-1 fresh soil did not influence severity of root and crown rot caused by Fusarium oxysporum f.sp. asparagi (Foa). Root residues accumulated in field soil during asparagus growing for 10 years did not influence disease severity either. Inoculation of this soil with laboratory-prepared Foa after treatment at 65°C (30min), at which the indigenous pathogen was killed but toxic substances present in asparagus root residues were left undamaged, led to the same disease severity as inoculation of similarly-treated fresh soil.
On soil extract agar, aqueous root extracts of asparagus but not those of other crops retarded growth of 31 out of 112 fungal isolates from a range of taxa. Sensitive fungi included Gliocladium spp. and Trichoderma harzianum , but not Foa.
Colonization of Foa-infested soil by Fusarium species was greatly enhanced by addition of root material from asparagus, Brussels sprouts, and chicory, but not by that from strawberry and perennial rye grass. As the fraction of Foa amongst the Fusarium population was small, it is concluded that competitive saprophytic ability of the pathogen is far less than that of the nonpathogenic Fusarium species. Fungistasis to Foa was not or was only slightly reduced in soils amended with root residues.
In contrast to data reported in the literature, the present results do not suggest an appreciable increase of Foa root rot., or of the Foa population in soils, due to substances present in root residues.     

Assessing mango anthracnose using a new three-dimensional image-analysis technique to quantify lesions on fruit

An accurate image-analysis method was developed to assess quantitatively the spot-like lesions on fruits resulting from pathogen attack. The technique was applied to evaluation of the development and severity of anthracnose of mango fruit, caused by the fungus Colletotrichum gloeosporioides . In this method, a stepper motor rotates the mango fruit along its longitudinal axis while acquiring a sequence of 360 images of its total surface (one image for each degree). This set of images is used to create a pseudocylindrical 'equal-area' projection of the fruit in a two-dimensional map containing complete morphometrical and photometrical information of its surface. The lesion area can easily be evaluated from this map with image-analysis procedures. Quantitative data (percentage of area affected) can be used to establish an assessment scale for the disease based on lesion spots measured, as well as for detailed laboratory studies of mango anthracnose development. The average error of the method is −0·1%, standard deviation 0·44 ( r ² = 0·99), and it may be adapted for use with most commercial image analysers and for other diseases with spot-like symptoms.     

Involvement of soilborne Phytophthora species in Central European oak decline and the effect of site factors on the disease

A survey was made on the occurrence of soilborne Phytophthora species in 35 oak stands on a range of geologically different sites in Bavaria. The most widespread species were P. quercina , P. cambivora and P. citricola . Seven other Phytophthora species were isolated infrequently. The fine root systems of 106 healthy and 111 declining mature trees of Quercus robur and Q. petraea were intensively investigated. The results indicate that, depending on the site conditions, at least two different complex diseases are referred to under the name 'oak decline'. On sites with a mean soil pH (CaCl₂)  3·5 and sandy-loamy to clayey soil texture Phytophthora spp. were commonly isolated from rhizosphere soil, and highly significant correlations existed between crown transparency and various root parameters. Oaks with P. quercina or other Phytophthora spp. in their rhizosphere had markedly higher levels of fine root damage than oaks without Phytophthora spp., and were subject to a relative risk of severe crown symptoms of 2·1 and 2·8, respectively. In contrast, in stands with sandy to sandy-loamy soils and a mean soil pH  3·9, Phytophthora spp. were not found. In these stands, correlations between crown transparency and various root parameters were either less significant or not significant. It is concluded that Phytophthora species are strongly involved in oak decline on sandy-loamy to clayey sites with a mean soil-pH (CaCl₂)  3·5.     

Real-time quantitative PCR assays for evaluation of soybean varieties for resistance to the stem and root rot pathogen Phytophthora sojae

Phytophthora root and stem rot caused by Phytophthora sojae is one of the most destructive disease of soybeans in the world. Effective management of the disease depends on selection and use of soybean varieties resistant to the disease. Fast and reliable procedures are vital to screen soybean varieties against the pathogen. Novel real-time quantitative (qPCR) assays were developed for both absolute and relative quantification of P. sojae in infected root tissues. QPCR assays were based on the detection of the internal transcribed spacer (ITS) gene of the pathogen and 18S ribosomal gene of the host plant. Absolute qPCR allowed the detection of as low as 10 femtograms (fg) of P. sojae DNA in soybean roots. Relative qPCR, employing the comparative threshold cycle (Ct) method, was effective and reliable for quantification of P. sojae DNA normalized to plant DNA in infected soybean root tissues. P. sojae DNA quantities detected in both qPCR assays had high correlations with disease severity index (DSI) ratings of soybean varieties. QPCR assays developed in this study were useful for determination of the levels of P. sojae DNA in different varieties of soybean and for evaluation of them for relative resistance to the pathogen.     

Survival of Leptosphaeria maculans in soil on residues of Brassica napus in South Australia

The survival of Leptosphaeria maculans , which causes phoma stem canker (blackleg), on oilseed rape residues ( Brassica napus ) in South Australia was investigated. Using a quantitative polymerase chain reaction (PCR) assay for L. maculans DNA, the pathogen was mainly detected in the upper 5 cm of the soil profile, including residues on the soil surface. As the size of organic matter particles in the soil decreased, so did the quantity of L. maculans detected in them. To obtain representative data for a field, at least 30 subsamples needed to be collected over the 0·81 ha area studied. In a survey of 49 commercial fields in South Australia, most L. maculans was detected in fields 1 year after oilseed rape had been grown, with less detected after 2 years and negligible amounts 3 years or more after cropping. The diagnostic DNA-based assay for L. maculans reduced the time and cost of studying L. maculans survival in soil and increased the sensitivity and accuracy of results compared with estimates of propagule number of colony-forming units on a semiselective medium.     

Variation among Colletotrichum gloeosporioides isolates from infected coffee berries at different locations in Vietnam

Isolates of Colletotrichum gloeosporioides associated with anthracnose disease on coffee berries in Vietnam were characterized by morphological and molecular methods. Random amplified polymorphic DNA (RAPD) and microsatellite-primered PCR (MP-PCR) analyses were employed to investigate the genetic variation among 38 and 51 isolates of C. gloeosporioides , respectively. According to both methods, the isolates mainly grouped in accordance with geographical origins. Higher genetic variation ( H  = 0·312 and 0·335) in the northern population of C. gloeosporioides than in the southern population ( H  = 0·261 and 0·186), according to the RAPD and MP-PCR markers, respectively, was indicative of a difference between the northern and southern populations. Moderate gene differentiation ( G st = 0·1) between populations from the north and the south was found. However, there was no differentiation between locations within the northern or southern populations, indicating significant gene flow. A four-gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam. The symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD (coffee berry disease; Colletotrichum kahawae ) isolates originating from Africa.     

Studies on the epidemiology and yield losses from rice black-streaked dwarf disease in a recent epidemic in Zhejiang province, China

The spread of rice black-streaked dwarf disease, which has emerged as a major problem on winter wheat and the two summer rice crops (early indica and late japonica ) grown in central and southern Zhejiang province, China, is documented from 1995 to 2007. The late japonica crop suffered the most: up to 64 640 ha were affected with estimated losses of c . 120 000 t grain per year. Peak adult numbers of the small brown planthopper vector, Laodelphax striatellus , coincided with the seedling stages of both rice crops and the proportion of the insect population carrying virus increased during 1998–2005. Seedlings with three to four leaves were the most susceptible, whereas plants inoculated after the end of tillering developed few or no symptoms. Disease levels were strongly correlated with numbers of viruliferous vectors. In sowing-date experiments with both rice crops, the earliest sowings had the most disease and suffered the greatest yield losses. With the last sowing date (25 days after the first), there were almost no losses. There were yield losses of 0·80% for every 1% increase in disease incidence in early indica rice and rather more (0·92%) in the late japonica crop. There were large differences in susceptibility between cultivars, indicating the possibility, within currently available germplasm, of using more resistant cultivars to help contain the disease. Changes in cropping practice and in recent winter weather conditions have probably contributed to the emergence of the virus as a major pathogen in eastern China.     

Factors affecting the incidence and severity of Spongospora subterranea infection and galling in potato roots

The incidence and severity of root infection and root galling caused by Spongospora subterranea were assessed in potato plants (cv. Estima) grown under controlled environmental conditions. The effects of temperature, soil type, soil moisture regime and soil inoculum level on infection and root gall development were determined by molecular and visual methods at two plant growth stages. Root gall severity was scored at harvest, after which DNA was extracted from the roots and quantified in a real-time polymerase chain reaction (PCR) assay specific for S. subterranea . Root galling was severe at 17°C, with a disease score of 3·1 on a 0–4 scale, low (0·6) at 12°C, and did not occur at 9°C. The level of inoculum in soil, in the form of artificially added sporosori, had no effect on the incidence and severity of visual symptoms, with 21%, 41% and 33% incidence observed at 5, 15 and 50 sporosori g⁻¹ soil, respectively. Incidence of infection, as detected by the real-time PCR assay, was greater with increasing soil inoculum concentrations, ranging from 48% at 5 sporosori g⁻¹ to 59% (15 sporosori g⁻¹) and 73% (50 sporosori g⁻¹) of plants infected at maturity, but this effect was not statistically significant. No correlation was found between the occurrence of galls on roots and powdery scab on tubers of the same plants.     

Leaf scald (Xanthomonas albilineans) incidence and its effect on yield in seven sugarcane cultivars in Guadeloupe

Seven 5—month-old sugarcane cultivars difTering in resistance to leaf scald disease were inoculated by the decapitation technique with Xanthomonas albilineans. The effects of disease progress and incidence on yields were studied for the plant (first harvest) and two ratoon crops (second and third harvest). The percentage of diseased stalks and disease severity at first harvest 5 months after inoculation were 0·7 and 0·4, respectively, for the most resistant cultivar and 71·0% and 63·3, respectively, for the most susceptible cultivar. They decreased in all cultivars in both ratoon crops, but were still important in one cultivar (B69379). Significant ( P = 0·05) yield reductions of 12% and 21% occurred in two of the seven cultivars (B69566 and B69379, respectively). The number of symptomatic sugarcane stalks in the first ratoon crop (second harvest) was lower than the number of stalks colonized by the pathogen. Symptoms and yield losses of cultivars R570 and B69566 varied with the crop. Yield losses occurred in cultivar R570 only in the plant crop when this cultivar displayed numerous symptoms. Cultivar B69566 appeared to recover from the disease to a certain extent from the plant to the second ratoon crop (third harvest), as did the resistant cultivars in the first ratoon crop. In contrast, severe leaf scald symptoms were observed in the case of cultivar B69379 regardless of the crop, and significant yield losses occurred in the two ratoon crops. These results support the recommendation that cultivar B69379 should not be replanted in Guadeloupe.     

Detection and quantification of Fusarium commune in host tissue and infested soil using real‐time PCR

Fusarium wilt caused by Fusarium commune is a major limiting factor for Chinese water chestnut (Eleocharis dulcis) production in China. A SYBR Green I real‐time quantitative polymerase chain reaction (qPCR) assay was developed based on the mitochondrial small subunit rDNA of F. commune. Assay specificity of the FO1/FO2 primer set was tested on 41 fungal isolates, and only a single PCR band of c. 178 bp from F. commune was amplified. The detection limits of the assay were 1 fg μL^?1 pure F. commune genomic DNA, 1 pg μL^?1 F. commune genomic DNA mixed with host plant genomic DNA (0·5 ng μL^?1), and 1000 conidia/g soil (artificially inoculated). The amount of F. commune DNA in stem tissues detected by qPCR was significantly correlated with the disease severity (DS) ratings; however, the qPCR assay showed no significant positive correlation between spore densities in soil of different fusarium wilt DS groupings and the DS ratings. The qPCR assay was further applied to 76 soil samples collected from commercial fields of E. dulcis during the 2011 and 2012 growing seasons. The spore density of F. commune detected was positively correlated with disease index in the 2012 growing season but not in 2011. The qPCR method can be used for rapid and specific detection of F. commune in plant and soil samples, which will facilitate monitoring of the pathogen and improvement of disease management.