Antagonistic effect of soil rhizosphere microorganisms on Bipolaris sorokiniana,the causal agent of wheat seedling blight

The biocontrol efficiency of Epicoccum purpurascens, Gliocladium roseum, three strains of Bacillus subtilis, and Pseudomonas fluorescens, isolated from the rhizosphere of wheat plants, was assessed in relation to seedling blight caused by Bipolaris sorokiniana. An in vitro study of the potential antagonist was performed using the dual culture technique and by ‘sowing’ wheat seeds pelleted with the saprophytes in plates with water agar?+?the pathogen. In vivo assays were carried out in the greenhouse and in the field with pelleted seeds sown in artificially infested soil. Both the number of living plants and the number of plants with necrosis on the leaves and the base of the stems and roots were assessed 15 days after sowing. Under greenhouse conditions, B. subtilis 3 and G. roseum reduced the level of infection of Buck Pucará and Trigomax 100 cultivars, respectively. In the field, biocontrol of the disease was not achieved.     

Tracing seed to seedling transmission of the wheat blast pathogen Magnaporthe oryzae pathotype Triticum

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT), initially restricted to South America, is a global threat for wheat after spreading to Asia in 2016 by the introduction of contaminated seeds, raising the question about transmission of the pathogen from seeds to seedlings, a process so far not well understood. We therefore studied the relationship between seed infection and disease symptoms on seedlings and adult plants. To accomplish this objective, we inoculated spikes of wheat cv. Apogee with a transgenic isolate (MoT-DsRed, with the addition of being resistant to hygromycin). We identified MoT-DsRed in experiments using hygromycin resistance for selection or by observation of DsRed fluorescence. The seeds from infected plants looked either apparently healthy or shrivelled. To evaluate the transmission, two experimental designs were chosen (blotter test and greenhouse) and MoT-DsRed was recovered from both. This revealed that MoT is able to colonize wheat seedlings from infected seeds under the ground. The favourable conditions of temperature and humidity allowed a high recovery rate of MoT from wheat shoots when grown in artificial media. Around 42 days after germination of infected seeds, MoT-DsRed could not be reisolated, indicating that fungal progression, at this time point, did not proceed systemically/endophytically. We hypothesize that spike infection might occur via spore dispersal from infected leaves rather than within the plant. Because MoT-DsRed was not only successfully reisolated from seed coats and germinating seeds with symptoms, but also from apparently healthy seeds, urgent attention is needed to minimize the risks of inadvertent dispersal of inoculum.     

Verticillium longisporum and V. dahliae: infection and disease in Brassica napus

Verticillium wilt of oilseed rape ( Brassica napus ) is caused primarily by Verticillium longisporum and has become a serious problem in northern Europe. In order to evaluate whether V. longisporum and V. dahliae differ in their interaction with oilseed rape, phenotypical and molecular assessments were made. Oilseed rape plants for fungal assessments were inoculated with V. longisporum and V. dahliae via root-dipping and samples were taken from roots, stems, leaves, flowers, pods and seeds during plant development. The infection by V. longisporum was found to start mainly in lateral roots and root-hairs, followed by colonization of the xylem vessels and extensive spread in stems and leaves, whereas V. dahliae infected the main roots and remained in the region below the cotyledon node of the plants. Re-isolation studies, together with PCR analysis of samples taken from early growth stages through to fully ripe plants, showed that the onset of flowering was a critical phase for V . longisporum to colonize plants. No seeds infected with V. longisporum were found. Mycelial growth from V. dahliae but not V. longisporum was significantly reduced on media containing tissue from a low glucosinolate B. napus genotype compared with growth on media containing tissue from a high glucosinolate cultivar. The results of this study suggest that V. longisporum favours B. napus as host and that the transition from the vegetative to the generative phase is of importance for the spread of the fungus in oilseed rape plants.     

Transmission of <Emphasis Type="Italic">Colletotrichum coccodes via</Emphasis> tomato seeds

Anthracnose of tomato caused by Colletotrichum coccodes is a devastating disease of ripe fruits. This pathogen may also infect tomato roots, stems and leaves. In the present study, C. coccodes is shown to be capable of contaminating seeds collected from artificially inoculated tomato fruits. Seedlings germinating from these infected seeds exhibited disease symptoms and therefore may transmit the pathogen to the next crop. The proportion of infected seeds ranged between 20% and 63% in all C. coccodes isolates tested and correlated with the aggressiveness of the isolates to tomato fruits. Fungicidal treatment of the collected seeds reduced, but did not eliminate, seed infection. A transgenic C. coccodes isolate expressing green fluorescent protein was used to visualize the pathogen. Mycelium was observed both on surfaces of the seed coat and within 1% of the embryos.     

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.     

<Emphasis Type="Italic">Erwinia aphidicola</Emphasis> isolated from commercial bean seeds (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

In late 2003, a new disease appeared in protected bean crops in southeastern Spain, causing a decrease of over 50% in production. Several samples of affected plants were collected and analyzed and the agent of this disease was identified as the bacterium Erwinia aphidicola, which had never been described as a pathogen previously. We attempted to determine the possible bacterium transmission through seeds, using 120 commercial bean seeds from the same batch as that used in an affected farm, and 120 seeds from the fruiting plants of the same farm. Seed coats, cotyledons and leaves of plants originating from them, were taken and analyzed. Several of the developed symptoms on plants from commercial and fruiting plant seeds were internervial chlorosis, necrotic pits and rough roots and they coincided with those observed on affected crops. Bacteria present in commercial seed cotyledons were isolated and analyzed by biochemical and molecular tests. Results confirmed the presence of Erwinia aphidicola in four analyzed seeds; moreover, Bacillus simplex/Bacillus muralis, Pseudomonas mendocina, Pseudomonas putida and Paenibacillus polymyxa were also identified.     

Infection and colonization of strawberry by <Emphasis Type="Italic">Gnomonia fragariae</Emphasis> strain expressing green fluorescent protein

Gnomonia fragariae is a poorly studied ascomycete, which was recently demonstrated to be a cause of severe root rot and petiole blight of strawberry. The pathogen was genetically transformed with the GFP as a vital marker and hygromycin resistance gene. Several stable transformants were obtained, which did not differ in their phenotype from the wild type isolate. Using one of the GFP-tagged isolates the infection process and colonization of roots and petioles of host plant by the pathogen were studied. Fluorescence microscopy examinations of the inoculated plants at different time points showed that plant infection occurs 24 h after inoculation and intensively continues during first 3 days. The specific penetration sites on epidermal cells and preferences in colonization for certain root and petiole tissues were observed. The pathogen intensively colonized and destroyed cortex of roots and petioles and spread rapidly longitudinally within intercellular spaces. The petioles were colonized by the hyphae, which grew mostly in the intracellular spaces of the cortical cells while in the roots the intracellular growth of hyphae occurred only in the later stages of infection. The fungus was also capable to infect the vascular tissues of petioles although these were not the primary tissues colonized by the pathogen. The mature ascomata were formed on the infected petiole bases several weeks after the inoculation. This study presents a genetic transformation method for Gnomonia fragariae and it demonstrates details on infection process and colonization of root, crown and petiole tissues of strawberry by the pathogen.     

Advanced lentil lines screened for resistance to <Emphasis Type="BoldItalic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="BoldItalic">lentis</Emphasis> under greenhouse and field conditions

Fusarium oxysporum f. sp. lentis is the most important pathogen of lentil plants, and most areas under lentil cultivation are reported to have a fusarium wilt disease background. The plants are infected in the seedling stage and later stages of their development. Fusarium wilt disease, which has appeared at high incidence rates during recent years, has caused sharp drops in the yield, especially in Moghan, in the northwest of Iran. Forty-five isolates of the pathogen were collected from different regions of the country with two isolates from ICARDA in the summer of 2008 and identified using Nelson’s key. The pathogenicity of the collected isolates was studied on a sensitive line (ILL 4605) under greenhouse conditions and significant differences in pathogenicity were found among them. The most pathogenic isolates from three provinces, East Azerbaijan (EA 30), Ardebil (Ar 3) and Khorasan (Kh 45), were selected and used in screening of 55 developed lines under greenhouse and field conditions. In the greenhouse, test plants were inoculated by immersing root tips in spore suspension and sowing seeds in pre-infested pot soil. Field tests were carried out in a naturally highly infested farm. At all stages, the plant response to the disease was based on the percentage of dead plants. Cluster analyses of the greenhouse and field data led to the selection of three lines (81S15, FLIP2007-42 L and FLIP2009-18 L) that were resistant under greenhouse and field conditions.     

Virus infection and reproductive losses in faba bean (Vicia faba L.)

The viruses, bean yellow mosaic ( BYMV ), Echtes Ackerbohnenmosaik (EAMV) and bean (pea) leaf roll ( BLRV ) reduced seed yield in faba bean particularly when plants were infected at the pre-and mid-bloom stage. EAMV and BYMV , but not BLRV , delayed senescence and increased branching on glasshouse-grown plants so that more inflorescences were produced on diseased plants; most of the additional flower buds necrosed. All three viruses increased the proportion of flower buds that became necrotic thus reducing the number of mature flowers available for pollination. This, together with enhanced abscission of recently set flowers, diminished pod production. Flower set per se was unaffected by infection. The number of ovule sites per pod and weight of individual mature seeds were also unaffected by these diseases but productivity per pod declined because of increased ovule abortions. Patterns of pod production on the inflorescence and location of mature seeds within the pod were unaffected by virus infection.     

Root rot pathogens in field soil,roots and seeds in relation to common bean (Phaseolus vulgaris), disease and seed production

The interrelationships among bean productivity, prevalence of pathogens in roots, seeds and soil, and root rot disease were described at the pod maturity stage in 13 commercial fields. The soil population and frequency of pathogens isolated from seeds varied by pathogen species and field location. Fusarium solani was the most prevalent fungus isolated from bean seeds and field soil compared to Rhizoctonia solani, Macrophomina phaseolina and F. oxysporum. Principal component analysis revealed that the first component explaining 32% of the total variance was correlated with the root rot index. PC1 was more strongly linked to root and seed infections in comparison with soil populations of pathogens. Based on a correlation between PC2 (accounting for 23% of the total variance) and the number of seeds per bean plant, charcoal, Fusarium and Rhizoctonia root rots were recognized as more important determinants of seed losses to root rot disease. There were correlations among the major pathogens infecting either roots or seeds of beans. These findings provide useful information for future experimental plans to optimize management strategies for bean root rots.     

Development of a powder formulation of Pseudomonas fluorescens for control of rice blast

Pseudomonas fluorescens strain Pf1, inhibitory to the growth of the rice blast pathogen Pyricularia oryzae in vitro , was developed as a talc-based powder formulation. When rice seeds were treated with this formulation, the bacteria spread to roots, stems and leaves of the plants and protected against leaf infection by P. oryzae . When applied as a foliar spray, the bacteria survived on the leaves. The powder formulation controlled leaf blast under greenhouse conditions. In tests as a seed treatment and foliar spray in four field trials it effectively controlled the disease and increased grain yield.     

Influence of plant stage and organ age on the receptivity of <Emphasis Type="BoldItalic">Pisum sativum</Emphasis> to <Emphasis Type="BoldItalic">Mycosphaerella pinodes</Emphasis>

On spring pea, ascochyta blight (Mycosphaerella pinodes) frequently appears at the plant base on yellowing stipules and disease scores are higher on basal parts of the plants than on the uppermost parts. In order to investigate the relationship between pea plant growth stage and/or organ age, and ascochyta blight on whole plants and detached stipules and pods, two experiments were conducted in 2009 and 2010 under controlled conditions. This study showed that plant stage does not influence receptivity to ascochyta blight before the appearance of the first visual signs of senescence (beginning of yellowing). When stipules were green, regardless of the plant stage, no differences of receptivity was observed (except for the youngest stipule(s) at the top of the plant). Plant stage only had an effect on receptivity by the way of the importance of the visual senescence of the organs and visual senescence has a more predominant effect on plant receptivity than the plant stage considered alone. An effect of the nodal position, linked with the stipule age, was observed on whole plant inoculation, with a decreasing disease severity gradient from the base to the top of the plant which was explained by a greater receptivity for the basal/older stipules which had begun yellowing. On detached stipules, a higher receptivity was observed as soon as visual senescence had been observed. On pods, during their filling, few symptoms appeared and severity was important as soon as they began to yellow. The effect of ascochyta blight on induced senescence of the infected stipules was also observed.     

Effects of soil moisture and sowing depth on the development of bean plants grown in sterile soil infested by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>

The effects of soil moisture (varying from 15% to 42% (v/v)) and sowing depth (1.5–6.0 cm) on the development of bean plants grown in sterile soil infested by the pathogen Rhizoctonia solani and its antagonist Trichoderma harzianum were studied under greenhouse conditions. The four possible combinations of soil infestation with both fungi were tested. Disease severity, percentage of plants emerged, plant height and dry weight were evaluated 3 weeks after sowing. Emergence rate and growth of plants inoculated only with R. solani were not affected by soil moisture, but in the presence of both fungi, plant emergence, plant height and dry weight significantly decreased when soil moisture diminished. Deep sowing significantly reduced the emergence rate and growth of those plants that were inoculated with R. solani only. However, when the soil was infested with both fungi, the effect of sowing depth was not significant. At a sowing depth of 6.0 cm, the percentage of plants emerged was 50% in the presence of T. harzianum, but only 6.7% when the pathogen was inoculated alone. The antagonist protected bean seedlings from pre-emergence damping-off, reduced disease severity and increased plant growth in the presence of R. solani, especially in moist soil.     

Cytomolecular aspects of rice sheath blight caused by <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Sheath blight, caused by anastomosis group 1-IA of Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris (Frank) Donk), is one of the most destructive rice diseases worldwide. The pathogen is able to infect plants belonging to more than 27 families, including many economically important monocots and dicots such as rice, wheat, alfalfa, bean, peanut, soybean, cucumber, papaya, corn, potato, tomato and sugar beet. It is a soil borne necrotrophic fungus that survives in plant debris as sclerotia, which are small brown-to-black, rocklike reproductive structures. The sclerotia can survive in the soil for several years and infect rice plants at the water-plant interface in the flooded field by producing mycelia. Management of rice sheath blight requires an integrated approach based on the knowledge of each stage of the disease and cytomolecular aspects of rice defence responses against R. solani. This review summarizes current knowledge on molecular aspects of R. solani pathogenicity, genetic structure of the pathogen populations, and the rice-R. solani interaction with emphasis on cellular and molecular defence components such as signal transduction pathways, various plant hormones, host defence genes and production of defence-related proteins involved in basal and induced resistance in rice against sheath blight disease.     

Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae , is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30-cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2-m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR-specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.     

Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post‐flowering stress. Overseas reports of M. phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperature × soil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M. phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130 mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M. phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.     

Infection of roots of Dieffenbachia maculata by the foliar blight and soft rot pathogen, Erwinia chrysanthemi

Erwinia chrysanthemi, the organism causing foliage blight, leaf spotting, basal stem rot and root rot of Dieffenbachia maculata, was shown to be capable of infecting via roots. Inoculated plants were sectioned and internal spread of the pathogen determined by 'sandwich' plating between two layers of Miller's selective medium. Light microscopy and SEM confirmed that the pathogen was present throughout the plant in the xylem. Resin ducts, previously claimed as being the route for systemic infection, were not found. Initially confined to the xylem, the pathogen spread intercellularly to the neighbouring parenchyma and, after about 8 days, pockets of infection, often surrounded by periderm–like tissues, were seen.     

麦类赤霉病菌在水稻上存活的进一步研究

 本文报道浙江省北部的麦-早稻-晚稻的三熟制地区,麦类赤霉病菌在麦收后能侵染早、晚稻的穗部和稻株基部叶鞘。经取样测定,8块田的平均穗感染率20.63%,谷粒感染率为5.51%,稻株基部感染率为19.38%。这一结果进一步证实翌年早春稻桩产生的玉米赤霉子囊壳,是在晚稻生长后期已被该菌侵染所致。除外,在水稻上还分离到弯角镰孢、同色镰孢和单阳镰孢。本试验采用小麦粒、琼脂培养基作为诱发镰孢属菌株产生分生孢子和禾谷镰孢的有性世代子囊壳,在菌株鉴定中有很好的实用价值。     

Colonisation and histological changes in muskmelon and autumn squash tissues infected by <Emphasis Type="Italic">Acremonium cucurbitacearum</Emphasis> or <Emphasis Type="Italic">Monosporascus cannonballus</Emphasis>

Muskmelon (Cucumis melo cv. Temprano Rochet) and autumn squash (Cucurbita maxima) seedlings were inoculated either with Acremonium cucurbitacearum or Monosporascus cannonballus, two of the soil-borne fungi implicated in ‘melon collapse’. Inoculation was achieved in two different ways: by growing the plants in pots containing infested soil to study the histological changes produced in the infected tissues using light microscopy and by growing seedlings in Petri dishes together with fungal colonies in order to observe the colonisation of the plant tissues using scanning electron microscopy. Both muskmelon and autumn squash roots infected with A. cucurbitacearum showed a suberised layer in the epidermis and the outermost layers of the parenchymatic cortex, but these symptoms developed earlier in the muskmelon plants. Muskmelon plants infected by this fungus also presented hypertrophy and hyperplasia, which led to a progressive separation of the vascular bundles in the lower stems of the affected plants. This response was not observed in autumn squash during the study. On the other hand, few histological changes were observed in tissues infected with M. cannonballus and only a slight increase in the size of cortical intercellular spaces was noted in the lower stems of muskmelon plants, and infected autumn squash tissues remained free of these symptoms throughout the study. The scanning electron microscope observations revealed that both fungi were able to colonise the tissues of the two host plants which were studied. A. cucurbitacearum colonised the epidermis and cortex of both muskmelon and autumn squash. The hyphae grew both inter- and intracellularly, and the density of the colonisation decreased within the endodermis. The same colonisation of host plants was observed as a result of M. cannonballus infection. The xylem vessel lumina of both muskmelon and autumn squash showed hyphae and tylose formation as a result of both fungal infections. However, non-fungal structures were detected in the hypocotyl vascular tissues. The present study demonstrates that both fungi are capable of infecting the tissues of a species which is resistant (autumn squash) and a species which is susceptible (muskmelon) to melon collapse.