Localized Melanization of Appressoria Is Required for Pathogenicity of Venturia inaequalis

ABSTRACT During formation of appressoria produced from conidia and ascospores of Venturia inaequalis, a dark brown ring structure was detected at the base of appressoria. This melanized appressorial ring structure (MARS) was attached to the leaf surface like a sealing ring and formed the fungus-plant interface; it is believed to be required for pathogen penetration of the cuticle. Neither germ tubes nor infection structures beneath the cuticle were found to be visibly melanized. MARS were formed not only on apple leaves but also on leaves of nonhost plants and artificial surfaces differing in hydrophobicity; the formation of appressoria and MARS was confined to hard surfaces. The melanin nature of the ring was confirmed by using melanin biosynthesis inhibitors. Applications prior to inoculation largely inhibited the melanization and reduced infection rate by 45 to 80%; curative applications were not effective. Transmission electron microscopy verified a localized melanization of the cell wall around the penetration pore, and melanin was incorporated into all layers of the fungal cell wall. Appressoria without MARS were not able to infect the plant, suggesting that this structure can be considered to be a pathogenicity factor in V. inaequalis.     

Automated identification of sugar beet diseases using smartphones

Cercospora leaf spot (CLS) poses a high economic risk to sugar beet production due to its potential to greatly reduce yield and quality. For successful integrated management of CLS, rapid and accurate identification of the disease is essential. Diagnosis on the basis of typical visual symptoms is often compromised by the inability to differentiate CLS symptoms from similar symptoms caused by other foliar pathogens of varying significance, or from abiotic stress. An automated detection and classification of CLS and other leaf diseases, enabling a reliable basis for decisions in disease control, would be an alternative to visual as well as molecular and serological methods. This paper presents an algorithm based on a RGB‐image database captured with smartphone cameras for the identification of sugar beet leaf diseases. This tool combines image acquisition and segmentation on the smartphone and advanced image data processing on a server, based on texture features using colour, intensity and gradient values. The diseases are classified using a support vector machine with radial basis function kernel. The algorithm is suitable for binary‐class and multi‐class classification approaches, i.e. the separation between diseased and non‐diseased, and the differentiation among leaf diseases and non‐infected tissue. The classification accuracy for the differentiation of CLS, ramularia leaf spot, phoma leaf spot, beet rust and bacterial blight was 82%, better than that of sugar beet experts classifying diseases from images. However, the technology has not been tested by practitioners. This tool can be adapted to other crops and their diseases and may contribute to improved decision‐making in integrated disease control.     

Thermographic assessment of scab disease on apple leaves

Phytopathogenic fungi may affect both the cuticular and the stomatal conductance of plant tissue resulting in significant modifications of leaf temperature. Venturia inaequalis colonizes apple leaves below the cuticle (subcuticularly) causing scab disease. The suitability of digital infrared thermography for sensing and quantifying apple scab was assessed by investigating the effects of V. inaequalis on the water balance of apple leaves in relation to the disease stage and the severity of scab. Transpiration was measured by infrared thermo-imaging to evaluate spatial heterogeneity of the leaves in response to localized infections. Fungal development was assessed microscopically. Subcuticular growth of the pathogen caused localized decreases in leaf temperature before symptoms appeared that significantly increased the maximum temperature difference (MTD) of leaves. The MTD increased with scab development and was strongly correlated to the size of infection sites (r2_linear = 0.85) and overall disease severity (% diseased leaf area, r2_square = 0.71). In later stages of the disease, the MTD decreased because of leaf senescence. Thermographic measurements revealed differences in disease severity resulting from disease stage, resistance of host tissue and differences in the aggressiveness of V. inaequalis isolates. Subcuticular growth of the pathogen was beyond the area of conidia production, therefore, the area of leaf with increased transpiration was larger than the scab lesions; the proportion decreased from >70% in the early stages to <20% for mature lesions. Leaf transpiration was increased by all stages of scab development, therefore, MTD may be used not only for the differentiation between diseased and non-diseased leaves, but also for disease quantification, e.g. in screening systems and monitoring in precision agriculture.     

Use of imaging spectroscopy to discriminate symptoms caused by Heterodera schachtii and Rhizoctonia solani on sugar beet

Diseases caused by nematodes and non-sporulating soil-borne fungi have low mobility and are likely to be suitable targets for precision agriculture applications. Sensors which assess the reflectance of plant leaves may be useful tools to detect soil-borne pathogens. The development of symptoms caused by the plant parasitic nematode Heterodera schachtii and the fungal pathogen Rhizoctonia solani anastomosis group 2-2IIIB alone or in combination was studied by leaf reflectance recorded with a hyperspectral imaging system (range 400–1000 nm) for 9 weeks twice per week. Three image processing methods were tested for their suitability to generate the most sensitive spectral information for disease detection. Nine spectral vegetation indices were calculated from spectra to correlate them to leaf symptom recordings. Supervised classification by spectral angle mapper was tested for the discrimination of leaf symptoms caused by the diseases. The symptoms of Rhizoctonia crown and root rot caused by R. solani and symptoms caused by H. schachtii induced modifications that could be detected by hyperspectral image analysis. Rhizoctonia crown and root rot symptom development in mixed inoculations was faster and more severe than in single inoculations, indicating complex interactions among fungus, nematode and plant. The results from this study under controlled conditions are currently used to transfer the sensor technology to the field.     

Spatial variability of fusarium head blight pathogens and associated mycotoxins in wheat crops

The spatial pattern of Fusarium‐infected kernels and their mycotoxin contamination was studied in four wheat fields in Germany using geo‐referenced sampling grids (12–15 × 20–30 m, 28–30 samples per field) at harvest. For each sample, frequency of Fusarium‐infected kernels and spectrum of species were assessed microbiologically; mycotoxin contents were determined by HPLC‐MS/MS analysis. Spatial variability of pathogens and mycotoxins was analysed using various parameters including Spatial Analysis by Distance IndicEs (sadie^® ). Microdochium majus, the most frequent head blight pathogen in 1998, was less frequent in 1999 and could not be detected in kernels from two fields in 2004. Fusarium avenaceum, F. graminearum and F. poae were the most frequent Fusarium species, with 7–8 species per field. The frequency of Fusarium‐infected kernels was 3–15% and the incidence of species showed considerable within‐field variability. Spatial patterns varied among Fusarium species as well as from field to field. Although pathogens and mycotoxin were often distributed randomly in the field, F. avenaceum, F. graminearum, F. poae, F. sporotrichioides, F. tricinctum and the mycotoxin moniliformin had an aggregated pattern in at least one field. Patterns are discussed in relation to spread of Fusarium species depending on inoculum sources, spore type, kind of dispersal, availability of susceptible host tissue and micro‐climate. Sampling of wheat fields for representative assessment of mycotoxins is complicated by random patterns of Fusarium‐infected kernels, especially where the frequency of infection is small.     

Localized adhesion of nongerminated Venturia inaequalis conidia to leaves and artificial surfaces

Adhesion to the host surface is the first step for successful plant pathogen development and has been reported to be associated with both passive and active processes. For conidia of Venturia inaequalis, which depend on leaf wetness for germination, this process has not yet been described. Conidia of V. inaequalis adhered to wet hydrophobic surfaces immediately after contact to the surface, hours before initiation of germination. Attachment of nongerminated conidia was much better on hydrophobic surfaces, such as apple leaves and polystyrene, than on hydrophilic glass. Conidia released adhesive material localized in a droplet named spore tip glue (STG) at the spore apex which interacted with a contact surface only when water was present. Histochemical investigations indicated the presence of proteins and carbohydrates in STG, lectin labeling the presence of beta-galactose and N-acetylglucosaminyl residues. Transmission electron microscopy revealed two phases in the STG at the tip of dry mature conidia; as STG was present on the outer side of the intact fungal cell wall its formation should be associated with the secretion of glue through pores of the conidial wall. Surface-active substances affected the adhesion of conidia to hydrophobic surfaces stressing the importance of hydrophobic interactions. The use of protein biosynthesis inhibitors did not affect adhesion of conidia indicating that the adhesive material was preformed. It is concluded that the coincidence of STG, contact to a hydrophobic surface, and free water are essential for the adhesion of V. inaequalis conidia.     

Mapping of the leaf rust resistance gene <Emphasis Type="Italic">Lr38</Emphasis> on wheat chromosome arm 6DL using SSR markers

Leaf rust caused by the fungus Puccinia triticina is one of the most important diseases of wheat (Triticum aestivum) worldwide. The use of resistant wheat cultivars is considered the most economical and environment-friendly approach in controlling the disease. The Lr38 gene, introgressed from Agropyron intermedium, confers a stable seedling and adult plant resistance against multiple isolates tested in Europe. In the present study, 94 F₂ plants resulting from a cross made between the resistant Thatcher-derived near-isogenic line (NIL) RL6097, and the susceptible Ethiopian wheat cultivar Kubsa were used to map the Thatcher Lr38 locus in wheat using simple sequence repeat (SSR) markers. Out of 54 markers tested, 15 SSRs were polymorphic between the two parents and subsequently genotyped in the population. The P. triticina isolate DZ7-24 (race FGJTJ), discriminating Lr38 resistant and susceptible plants, was used to inoculate seedlings of the two parents and the segregating population. The SSR markers Xwmc773 and Xbarc273 flanked the Lr38 locus at a distance of 6.1 and 7.9 cM, respectively, to the proximal end of wheat chromosome arm 6DL. The SSR markers Xcfd5 and Xcfd60 both flanked the locus at a distance of 22.1 cM to the distal end of 6DL. In future, these SSR markers can be used by wheat breeders and pathologists for marker assisted selection (MAS) of Lr38-mediated leaf rust resistance in wheat.     

Remote sensing to detect plant stress induced by Heterodera schachtii and Rhizoctonia solani in sugar beet fields

The characteristically clustered occurrence and low level of mobility of Heterodera schachtii and Rhizoctonia solani in the soil and the induction of stress symptoms in the sugar beet canopy make them ideal targets for site-specific arrangements with precision agriculture tools. A field site infested with H. schachtii and R. solani was investigated in 2009 with near-range and aerial hyperspectral sensors during the growing season. At 31 sample points ground truth data for incidence and severity of the two organisms were collected and geo-referenced. Spectral vegetation indices computed from reflectance measurements obtained from two flight campaigns (AISA, 17th of June; HyMap, 28th of August) and the near-range spectroradiometers were significantly correlated (P < 0.01) with symptoms caused by the nematode or Rhizoctonia crown and root rot. A supervised classification with Spectral Angle Mapper of leaf symptoms induced by the organisms resulted in a classification accuracy of 72 and 64% for the AISA and HyMap data, respectively. The results demonstrated that remote sensing in combination with geographic information system technologies can be used effectively for the detection and mapping of symptoms caused by beet cyst nematode and Rhizoctonia crown and root rot.     

Effect of fungicides on the complex of <Emphasis Type="Italic">Fusarium</Emphasis> species and saprophytic fungi colonizing wheat kernels

Fusarium head blight of wheat, often associated with mycotoxin contamination of food and feed is caused by various Fusarium species. The efficacy of fungicide sprays for the control of the disease and mycotoxins varies from being highly effective to even increasing mycotoxin levels. The potential role of antagonistic fungi in this variability was investigated assessing sensitivity of Fusarium species and saprophytic fungi colonizing wheat kernels to fungicides. Saprophytes were tested for their antagonistic activity to the prevalent Fusarium species Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, and Fusarium poae. Fungal isolates from mature winter wheat kernels were Alternaria alternata, Arthrinium sp., Aspergillus niger, Epicoccum sp., Microdochium spp., Rhizopus oryzae and Trichoderma sp. In dual culture A. niger, R. oryzae and Trichoderma hamatum were more effective in reducing mycelial growth of Fusarium species than Microdochium majus; A. alternata and Epicoccum sp. were ineffective because of slow growth rates. Saprophytic fungi were sensitive to triazoles; however, prothioconazole and tebuconazole had stronger effects on mycelial growth of Fusarium species. ED₅₀ values also indicated significant differences in the sensitivity of Fusarium species to triazoles (range 0.1–1.7 mg l⁻¹). Azoxystrobin and fluoxastrobin were largely ineffective in inhibiting in vitro growth of Fusarium spp.; sensitivity of the other fungi was generally lower, except for M. majus which was highly sensitive. Due to differences in fungicide sensitivity among Fusarium spp. and ear-colonizing fungi antagonistic to Fusarium spp. fungicides are likely to modify the balance within the mycoflora of wheat ears which may also affect the mycotoxin contamination of grain.