Brachypodium distachyon exhibits compatible interactions with Oculimacula spp. and Ramularia collo‐cygni,providing the first pathosystem model to study eyespot and ramularia leaf spot diseases

Brachypodium distachyon (Bd) has established itself as an essential tool for comparative genomic studies in cereals and increasing attention is being paid to its potential as a model pathosystem. Eyespot and ramularia leaf spot (RLS) are important diseases of wheat, barley and other small‐grain cereals for which very little is known about the mechanisms of host resistance despite urgent requirements for plant breeders to develop resistant varieties. This work aimed to test the compatibility of interaction of two Bd accessions with the cereal pathogens Oculimacula spp. and Ramularia collo‐cygni, the causal agents of eyespot and RLS diseases, respectively. Results showed that both Bd accessions developed symptoms similar to those on the natural host for all pathogen species tested. Microscopy images demonstrated that R. collo‐cygni produced secondary conidia and both Oculimacula spp. formed characteristic infection structures on successive tissue layers. Visual disease assessment revealed that quantitative differences in disease severity exist between the two Bd accessions. The results presented here provide the first evidence that Bd is compatible with the main causal agents of eyespot and RLS diseases, and suggest that future functional genetic studies can be undertaken to investigate the mechanisms of eyespot and RLS disease resistance using Bd.     

Characterization of DMI,QoI and SDHI fungicides sensitivity of Ramularia collo-cygni isolates in Argentina

Journal of Plant Diseases and Protection - Ramularia leaf spot (RLS) caused by the fungus Ramularia collo-cygni (Rcc) has become a threat to barley production in Argentina. All barley varieties are...     

Evidence for seed transmission and symptomless growth of Ramularia collo‐cygni in barley (Hordeum vulgare)

Ramularia collo‐cygni (Rcc) is becoming an increasing problem for barley growers across Europe. However, the life cycle of the pathogen is only slowly being elucidated. In this study, Rcc DNA was detected in a number of harvested seed samples from 1999 to 2010, with mean levels peaking in winter barley samples in 2009. A number of experiments were carried out to determine whether the pathogen could move from barley seed to seedlings, and also from seed through the developing plant and into the subsequent generation of seed, both in controlled experiments and in field trials. Results from testing of seed indicated that the fungus is widespread at the end of the growing season in harvested grain samples and can be transmitted to developing plants from infected seed stock. Examination of infected seedlings did not reveal the presence of spores but fungal structures were found within the leaf. The location of the fungus within seed was examined, with Rcc DNA found in both embryo and non‐embryo tissue. The implications for barley production of the pathogen being seedborne are discussed.     

A method of inoculating barley seedlings with Ramularia collo‐cygni

A method of inoculating seedlings with the fungus Ramularia collo-cygni, the causal agent of ramularia leaf spot (RLS), an increasingly important problem in barley in Europe and elsewhere, is described. Symptoms of RLS similar to those found in the field were reproduced on seedlings and the fungus was re-isolated from them, fulfilling the third and fourth of Koch's postulates. The method was similar to one used for the related fungus, Mycosphaerella graminicola (anamorph Septoria tritici), a pathogen of wheat. Briefly, plants were sprayed with a suspension of R. collo-cygni mycelium fragments, incubated at 15°C, first in darkness for 48 h then in a 16-h-light/8-h-dark cycle. Disease levels reached saturation when plants were sprayed to runoff with a suspension of 480 cm² of mycelium, scraped from the entire surface of 7·5 Petri dishes (9 cm diameter) and sieved, in 50 mL water. Growth of seedlings in high light intensity (900 µmol m⁻² s⁻¹, 16-h daylength) before inoculation increased disease symptoms, but reduced disease when applied after inoculation. In contrast to M. graminicola, near-ultraviolet light after inoculation reduced symptom development. It is proposed that for the full development of RLS, plants should be grown in a stressful environment before inoculation. Nine barley lines were assessed for their resistance to RLS as seedlings and a subset were tested in field trials with natural infection by R. collo-cygni. There was cultivar-by-isolate interaction in the amount of RLS symptoms on seedlings. RLS levels on adult plants in field plots were correlated with RLS scores on seedlings formed by one isolate but not the other.     

Exploring de novo specificity: the Pyrenophora tritici‐repentis–barley interaction

Pyrenophora tritici‐repentis (Ptr) is a destructive fungal pathogen of wheat worldwide. In addition to wheat, Ptr has been isolated from various other hosts in the family Poaceae, yet the nature of its interaction with those hosts is unknown. The Ptr–barley relationship was explored and the existence of a specific interaction between Ptr and barley is described for the first time; symptom development on several barley genotypes was evaluated in bioassays and by toxin infiltration into barley leaves. Ptr ToxB‐producing isolates of the fungus were able to cause significant damage when inoculated onto certain barley genotypes, and Ptr ToxB was able to induce chlorosis in a highly selective manner when infiltrated into those same genotypes. Ptr–barley specificity is subtle and can break with slight changes in temperature after infection. To understand the infection process in barley, a cytological analysis and in planta fungal biomass estimation using quantitative PCR were performed. The fungus penetrates through the host epidermal cells and advances to colonize the mesophyll layer intercellularly, with the infection process on barley closely resembling that on wheat. Here, evidence is provided for a specific interaction between barley and Ptr, expanding understanding of Ptr host specificity and breaking the assumption that the highest level of specificity seen with Ptr is restricted to particular genotypes of the wheat host.     

Ramularia leaf spot disease of barley is highly host genotype-dependent and suppressed by continuous drought stress in the field

Journal of Plant Diseases and Protection - Since the 1980s, Ramularia leaf spot (RLS) is an emerging barley disease worldwide. The control of RLS is increasingly aggravated by a recent decline in...     

A hypothesis on physiological alterations during plant ontogenesis governing susceptibility of winter barley to ramularia leaf spot

The occurrence and epidemic spread of ramularia leaf spot (RLS) caused by Ramularia collo-cygni was studied with relation to the plant age-dependent alterations in the antioxidative defence systems in the leaves during mature stages of field-grown winter barley. The breakdown of enzymatic activities of dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase in the two uppermost fully expanded leaf layers correlated well with a decrease in the pool of the non-enzymatic antioxidants ascorbate and glutathione. The general decline in the antioxidative systems occurred between 31 May and 8 June after ear emergence stages and preceded the first visible symptoms of RLS. The activities of peroxidases corresponded to an increase in phenolics and lignification which seemed to enhance the infectivity of the fungus rather than to protect the plant. The first R. collo-cygni conidia were trapped in the field about 4 weeks before the latent infection was first detected by ELISA, and 5 weeks before the first disease symptoms became visible. Both the counts of airborne conidia and the weather data recorded between April and June suggest that neither inoculum nor the weather conditions are limiting factors for infection by R. collo-cygni at earlier growth stages of barley. It is concluded that the relatively late development of RLS in the field in mid June is governed by the significant degradation of the antioxidative protection systems in the leaves of barley at the onset of ripening stages, rather than by environmental factors or the availability of airborne inoculum.     

Extracellular Matrix, Esterase and the Phytotoxin Prehelminthosporol in Infection of Barley Leaves by Bipolaris sorokiniana

Light microscopy and cryo-scanning electron microscopy showed that hyphae of Bipolaris sorokiniana adhered to the wax surfaces of barley leaves by means of an extensive extracellular matrix (ECM). Prehelminthosporol, the major non-host specific phytotoxin formed by B. sorokiniana was immunolocalized in large amounts in the ECM surrounding the hyphae. Similarly, esterase activity involved in degradation of the cuticular wax surface was found in the ECM. Therefore, it appears that the ECM is not only important for adhesion of the fungus to its host, but also functions as a sink of phytotoxins and lytic enzymes important for infection of the host plant.     

Differential behaviour of sheath blight pathogen Rhizoctonia solani in tolerant and susceptible rice varieties before and during infection

This study characterized the early infection and establishment of the sheath blight pathogen Rhizoctonia solani on a tolerant rice variety, Swarnadhaan (IET 5656), and a susceptible variety, Swarna (MTU 7029). Assays using whole plants showed that disease severity was higher in Swarna than Swarnadhaan. In a detached leaf assay, Swarnadhaan showed a disease index that was 50% less than that with Swarna. Rhizoctonia solani exhibited different growth behaviour in the tolerant and susceptible varieties. The pathogen showed more hyphal growth in the susceptible host than in the tolerant variety. It also showed profuse branching, making intimate contact with the host surface to form more inter‐ and intracellular structures, and greater sclerotial development in the susceptible host compared to the tolerant one. Using light and scanning electron microscopy, it was observed for the first time that the pathogen could intercept host surface structures and use these for anchorage or penetration. Transformed R. solani, expressing green fluorescent protein, was observed using confocal laser scanning microscopy to investigate pathogen behaviour, including the formation of infection cushions and subsequent colonization of the host tissues. This is the first ultrastructural report to characterize the differential behaviour of the sheath blight pathogen in the vicinity and within tolerant and susceptible rice plants.     

High levels of genetic and genotypic diversity in field populations of the barley pathogen Ramularia collo-cygni

The ascomycete pathogen Ramularia collo-cygni causes Ramularia leaf spot (RLS) on barley. Although R. collo-cygni is considerd an emerging disease of barley, little is known about genetic diversity or population genetic structure of this pathogen. We applied a set of polymorphic AFLP (Amplified Fragment Length Polymorphism) markers to investigate population genetic structure in two Northern European populations of R. collo-cygni. The distribution of AFLP alleles revealed low levels of population subdivision and high levels of genetic diversity at both locations. Our analyses included 87 isolates and of these 84 showed a unique genotype pattern. The genetic structure of populations in Scotland and Denmark is highly similar and we find no evidence of population sub-division. An analysis of molecular variance was used to show that 86 % of the variance is attributable to within field genetic variance. In spite of the high levels of genetic and genotypic diversity in the R. collo-cygni populations, we find significant evidence of linkage disequilibrium among the AFLP alleles using a multilocus analysis. We propose that the high levels of genotypic diversity and the lack of population differentiation result from considerable levels of gene flow between populations most likely mediated by seed borne dispersal of inoculum.     

Photodynamic oxygen activation by rubellin D,a phytotoxin produced by Ramularia collo-cygni (Sutton et Waller)

Ramularia collo-cygni, a pathogenic deuteromycete, is the causal agent of Ramularia leaf spot disease on barley. It produces a number of coloured metabolites of which one was identified as rubellin D, a phytotoxin only described for Mycosphaerella rubella until now. Rubellin D induces light- and concentration-dependent necrosis on barley leaves. In a biochemical model system we could demonstrate that rubellin D exhibits photodynamic activity triggering the light-dependent production of reactive oxygen species (ROS). The use of inhibitors of ROS production confirmed mainly the production of singlet oxygen and to a lesser extent superoxide. In addition, light-dependent peroxidation of α-linolenic acid by rubellin D was shown. Based on our results we present a model for ROS-mediated symptom induction on barley leaves by Ramularia collo-cygni.     

Most Colletotrichum species associated with tree tomato (Solanum betaceum) and mango (Mangifera indica) crops are not host‐specific

An important constraint for crop production in Colombia is the high incidence of anthracnose caused by Colletotrichum species. Although several studies have focused on these fungi, the relationship between the different fungal species within the genus and their hosts and whether they display any host preference or host specificity has yet to be examined. In Colombia, diseases caused by Colletotrichum species are particularly severe in mango (Mangifera indica) and tree tomato (Solanum betaceum) crops. In a previous investigation, the Colletotrichum phylogenetic species attacking these crops were identified. The present study aimed to determine whether isolates collected from tree tomato and mango showed host preference or host specificity by assessing aggressiveness, spore density, latent period, and fitness of each strain on the two hosts. In the departments of Cundinamarca and Tolima, Colombia, isolates were collected from plants that presented typical anthracnose symptoms and were identified as C. acutatum, C. asianum, C. boninense, C. gloeosporioides, C. tamarilloi and C. theobromicola. Inoculation of conidia of each isolate onto both hosts showed isolates had no host preference and only the C. gloeosporioides isolate showed host specificity. However, in general, isolates produced a higher spore density when inoculated on the alternate host, which may indicate a difference in the degree of adaptation to each host. Statistical analyses of the assessed parameter values revealed that isolates use different infection strategies when infecting each host. In light of these results, the implications of using quantitative estimations of fitness when studying fungal pathogens are discussed.     

Host Range of Tropical and Sub-tropical Isolates of Polymyxa graminis

The host range of Polymyxa graminis isolates originating from peanut clump-infested areas in India (Andhra Pradesh and Rajasthan), Pakistan and Senegal was studied on monocotyledonous and dicotyledonous cultivated species, using known quantities of sporosori as inoculum. Profuse multiplication occurred only on some graminaceous species, but the various isolates showed different host specificity. All the isolates produced high infection on sorghum and pearl millet, and all but one isolate from Rajasthan infected maize. Wheat, rye and barley were susceptible to some of the tested isolates. The isolates from Rajasthan and Pakistan produced moderate to severe infection on at least one of these species. On rice, groundnut and sugar beet, only traces of infection by some isolates were detected, whereas no infection was observed on mustard and sunflower. Differences of susceptibility in Pennisetum spp. and Sorghum spp. were demonstrated. The variations in host specificity among isolates from peanut clump-infested areas may result from an adaptation of P. graminis populations to various biotopes. The implications of these results for the management of peanut clump disease are discussed. A comparison of the host ranges of isolates of P. graminis and P. betae from temperate areas demonstrated that distinct types of Polymyxa might be identified based on their relative ability to multiply on susceptible species. Nevertheless, overlapping in the host ranges among the different Polymyxa types, characterised by distinct ecological and genomic features, raises doubts about the host range as a classification criterion for the Polymyxa genus.     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Histological methods to detect the clubroot pathogen Plasmodiophora brassicae during its complex life cycle

The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that lives in close relationship with its host cell. The roots of the host plants are colonized and the plant growth is altered upon infection. While shoots can be stunted and show wilt symptoms after longer infection periods, the root system is converted to a tumorous root tissue, called ‘clubroot’, by alterations of the plant growth promoting hormones auxin, cytokinin and brassinosteroid. Because the life cycle occurs largely within the host cells, this leads to dramatic changes in host root morphology and anatomy. Thus, the identification of the respective protist structures in the host tissue by microscopy is challenging. Different staining methods as well as fluorescence and electron microscopy of thin sections can reveal specific life stages of P. brassicae and can yield additional information on the changes in the host tissues concerning, for example, cell wall properties. In addition, promoter–reporter fusions, immunostaining methods and in situ hybridization techniques can be used to gain additional information on the changes in the host roots.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Testing and modelling the potential of three diploid plants in Poaceae as a new pathosystem to investigate the interactions between cereal hosts and cereal cyst nematode (Heterodera avenae)

Cereal cyst nematode (CCN), Heterodera avenae, is one of the most important pathogens of wheat worldwide, and causes significant yield losses. Research on CCN–wheat interactions is hampered by the lack of an effective model pathosystem. This study investigated the potential of the model cereal Brachypodium distachyon (Bd21‐3) and diploid wheat 2A (G1812) and 2D (AL8/78) as model hosts for CCN. Nematode infection analysis showed that although some CCN penetrated Bd21‐3 roots, these nematodes failed to develop to the later developmental stages or form cysts, indicating B. distachyon is not a host for CCN. A strong burst of reactive oxygen species (ROS) within Bd21‐3 roots infected with CCN was induced 3 days after infection and the expression of seven ROS‐producing genes was significantly increased. In contrast, CCN completed its life cycle in both diploid wheat 2A and 2D, and formed normal syncytia in these hosts. Although CCN developmental processes within both diploid wheat 2A and 2D were very similar to those in the susceptible control, the number of cysts formed on diploid wheat 2D was less than those formed on diploid wheat 2A and the susceptible control, indicating that diploid wheat 2A was a more suitable host for CCN than 2D. This is the first report of a potential new pathosystem for CCN–host interactions using diploid wheat.     

Susceptibility evaluation of Picea abies and Cupressus lusitanica to the pine wood nematode (Bursaphelenchus xylophilus)

Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28 days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well‐known PWN host. Furthermore, the stem ultrastructure of P. abies, C. lusitanica and Pn. pinaster was analysed by scanning electron microscopy. The results suggest that P. abies and C. lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P. abies seems to be a compatible host that could act as a repository for PWN.