Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

The role of the seed tuber in determining partial resistance to potato blackleg caused byErwinia spp.

In 1991 and 1992, 12 potato cultivars were screened at two locations for resistance to blackleg, after vacuum infiltration of the seed withErwinia carotovora subsp.atroseptica orE. chrysanthemi. Cultivar differences for resistance toE.c. subsp.Atroseptica andE. chrysanthemi were found which were consistent over locations and years. Seed tubers of the same cultivars were also screened for resistance to bothErwinia spp. by using a tuber slice inoculation method. Correlation coefficients for comparisons between resistance to blackleg in the field and tuber tissue resistance under aerobic or anaerobic conditions were not significant. This could partly be explained by drastic changes in relative tuber tissue resistance of the cultivars within a 5 weeks period after planting in the field. Presprouting of seed tubers in diffuse daylight had a less pronounced effect on relative tuber tissue resistance than planting in the field. Monitoring the process of mother tuber decay during the growing season of 1993 after vacuum infiltration withE.c. subsp.atroseptica andE. chrysanthemi revealed that cultivars differed in the extent to which these bacteria enhanced the process of mother tuber decay. These differences partly explained the cultivar differences for resistance to blackleg in the field.Abbreviations Eca Erwinia carotovora subsp.atroseptica - Ech Erwinia chrysanthemi - NOP Noordoostpolder - Wag Wageningen     

Identification of a new pathotype of Puccinia hordei with virulence for the resistance gene Rph7

Barley leaf rust resistance gene Rph7, derived from barley accession Cebada Capa, is the most effective R-gene for resistance to Puccinia hordei. Virulence for this gene was known in the USA, Israel and Morocco but not yet in Europe. We found an unexpected leaf rust infection in the field at Córdoba, Spain in 2004 on Rph7 carrying lines. This virulence for Rph7 was confirmed in growth chamber experiments, being the first report of Rph7 virulence in European populations of P. hordei. A collection of 680 barley accessions was screened for resistance against this new isolate. Twelve accessions showed segregation with individual plants showing resistance based on hypersensitivity (low infection type). These individual resistant plants were selected and grown in the greenhouse to obtain seeds.     

A comparative assessment of potential components of partial disease resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight using a detached leaf assay of wheat,barley and oats

The relative resistance of 15 winter barley, three winter wheat and three winter oat cultivars on the UK recommended list 2003 and two spring wheat cultivars on the Irish 2003 recommended list were evaluated using Microdochium nivale in detached leaf assays to further understand components of partial disease resistance (PDR) and Fusarium head blight (FHB) resistance across cereal species. Barley cultivars showed incubation periods comparable to, and latent periods longer than the most FHB resistant Irish and UK wheat cultivars evaluated. In addition, lesions on barley differed from those on wheat as they were not visibly chlorotic when placed over a light box until sporulation occurred, in contrast to wheat cultivars where chlorosis of the infected area occurred when lesions first developed. The pattern of delayed chlorosis of the infected leaf tissue and longer latent periods indicate that resistances are expressed in barley after the incubation period is observed, and that these temporarily arrest the development of mycelium and sporulation. Incubation periods were longer for oats compared to barley or wheat cultivars. However, oat cultivars differed from both wheat and barley in that mycelial growth was observed before obvious tissue damage was detected under macroscopic examination, indicating tolerance of infection rather than inhibition of pathogen development, and morphology of sporodochia differed, appearing less well developed and being much less abundant. Longer latent periods have previously been related to greater FHB resistance in wheat. The present results suggest the longer latent periods of barley and oat cultivars, than wheat, are likely to play a role in overall FHB resistance if under the same genetic control as PDR components expressed in the head. However the limited range of incubation and latent periods observed within barley and oat cultivars evaluated was in contrast with wheat where incubation and latent periods were shorter and more variable among genotypes. The significance of the various combinations of PDR components detected in the detached leaf assay as components of FHB resistance in each crop requires further investigation, particularly with regard to the apparent tolerance of infection in oats and necrosis in barley, after the incubation period is observed, associated with retardation of mycelial growth and sporulation.     

Susceptibility of Broccoli Cultivars to Bacterial Head Rot: In Vitro Screening and the Role of Head Morphology in Resistance

Head rot is a major disease of broccoli caused by the soft rot pathogens Pseudomonas fluorescens and Erwinia carotovora. Two in vitro pathogenicity tests were evaluated as a methods to identify broccoli cultivars susceptible or resistant to bacterial head rot. One test used mature heads excised from the plant and inoculated with squares of cotton lint which had been soaked in a bacterial suspension. The other test involved stab-inoculating axenically grown seedlings. With the excised head test, susceptible cultivars showed a black soft rot, whilst less susceptible or moderately resistant cultivars showed only watersoaking, or browning and slight softening of the tissue. No cultivar was completely resistant. Ten cultivars were tested, and their susceptibility ratings corresponded with previously recorded field data, with one exception. This laboratory test could be used to screen for susceptibility to head rot in broccoli breeding programmes. The seedling test distinguished differences in aggressiveness among bacterial isolates but not cultivar susceptibility. Increasing head size correlated negatively with disease resistance. Head shape, i.e. cultivars which showed a domed shape rather than a flat shape, was positively correlated with disease resistance. Thus small domed heads are more resistant to head rot than large flat heads. Other morphological characteristics, viz. floret prominence and number, and sepal stomatal number were not correlated with host resistance.     

Regional and varietal differences in the risk of wheat seed infection by fungal species associated with fusarium head blight in Italy

The incidence of seed infection by fungal species pertinent to the fusarium head blight complex was monitored from 1999 to 2002 in two soft and three durum wheat cultivars grown across the northern, central and southern production zones of Italy, in order to characterize the species composition at the seed level. The main species recovered were Fusarium graminearum, F. poae and Microdochium nivale. There was a marked influence of production zone on seed infection incidence for both durum and soft wheat cultivars, with incidence of infection generally decreasing from the northern to the southern zone. Incidence of seed infection by different species of Fusarium was twice to four times higher in durum compared with the soft wheat cultivars in the study. There were no significant differences in terms of seed infection incidence between the two soft wheat cultivars, but the durum cultivars differed in their levels of seed infection for some of the pathogens. The results demonstrated that the durum cultivars were more at risk of seed infection by pathogens associated with fusarium head blight, and that wheat grown in northern Italy is at higher risk of seed infection by these species.     

Differential interactions ofPhytophthora capsici isolates with pepper genotypes at various plant growth stages

Pepper cultivars from diverse geographic origins were evaluated for resistance to different isolates ofPhytophthora capsici under controlled environmental conditions. All accessions tested were susceptible at the four-leaf stage to the six isolates ofP. capsici. Inoculation at the eight-leaf stage resulted in significantly different interactions among the accessions andP. capsici isolates. The Korean and U.S. cultivars tested were highly susceptible to the isolates ofP. capsici at this stage. In contrast, PI 201234 and PI 201238 had a differential interaction with someP. capsici isolates. At the twelve-leaf stage, Phytophthora blight developed slowly in the Korean and U.S. cultivars that were highly susceptible at the eight-leaf stage. Furthermore, the accessions from the Asian Vegetable Research and Development Center (AVRDC) became highly resistant toP. capsici at this stage.     

Root hair infection by Plasmodiophora brassicae in clubrootresistant and susceptible genotypes of Brassica oleracea,B. rapa and B. napus

The pathogenesis of clubroot, a disease of cruciferous crops caused by the fungusPlasmodiophora brassicae, starts with infection of the root hairs. This process was studied in 13 accessions ofBrassica oleracea, B. napus and B. rapa with varying levels of plant resitance toP. brassicae. Seedlings were grown in a mineral solution, inoculated with resting spores ofP. brassicae, and the number of plasmodia developing in root hairs was recorded. When compared with the standard susceptible cultivar Septa, both higher and lower resistance to root hair infection was found in the accessions of the differentBrassica species. No complete resistance to root hair infection was found. Over the accessions studied, there was no correlation between the plant resistance estimated from greenhouse tests and the resistance to root hair infection of seedlings. The resistance of all accessions must at least partly be caused by other mechanisms which operate after the root hair plasmodia are formed.     

The role of seedling infection in epiphytotics of ascochyta blight on chickpea

Didymella rabiei, the causal agent of ascochyta blight, survives on infected seeds and seedlings. Diseased seedlings originating from infected seeds occasionally serve as the source for primary infection in chickpea crops. Experiments carried out independently in Australia and in Israel provided quantitative information on the temporal and spatial distribution of ascochyta blight from initial infections and on the relationship between the amount of initial infection and the intensity of subsequent epiphytotics for cultivars differing in susceptibility to the pathogen. Disease spread over short distances (<10 m) from individual primary infections, was governed by rain and wind, and was up to five times greater down-wind than up-wind. Cultivar response to D. rabiei significantly affected the distance and area over which disease spread and the intensity of the disease on infected plants. At onset of the epiphytotic, the relationship between disease spread and time was exponential (P < 0.05; R ² > 0.95) and the area of the resulting foci was over 10 times greater in susceptible cultivars than in resistant cultivars. Regression equations showed the relationship between disease severity and the distance from the focus-plants was inverse-linear for all cultivars tested (P < 0.05). A simulation model based on the experimental data revealed that even if primary infection is infrequent (less than 1% of plants), the consequences are potentially devastating when susceptible cultivars are used. The epidemiological information and simulation model generated by this study provide an increased understanding of the development of an epiphytotic in which the primary foci of disease originate from infected chickpea seedlings.     

Differences in resistance in onion cultivars to pink root rot disease in Iran

Forty-five onion (Allium cepa) cultivars were evaluated for disease resistance to the fungal pathogen Pyrenochaeta terrestris (pink root disease). Forty-three Iranian cultivars and two commercially exotic resistant cultivars (Texas Early Grano and Yellow Sweet Spanish) were grown in a glasshouse for two successive years (2003 and 2004) in Isfahan, Iran. Susceptibility of the cultivars was determined using a scale of 0–3. Six cultivars had significantly lower susceptibility of pink root compared to the exotic ones. High susceptibility tended to be associated with high mean scores (2–3), and the highly resistant cultivars had the lowest scores (0–1). In addition, the 45 examined cultivars were ranked from 1 to 45 according to their markedly differing reactions to P. terrestris, which differed markedly. Of 32 cultivars that were less resistant, 30 with an intermediate status or tolerance (1–2) differed considerably in their rank order. No pink root symptoms were seen in the early stages, but on blotter paper or wheat-straw agar some roots turned pink with no pycnidia formation, whereas on potato dextrose agar medium (PDA) with a few fresh healthy and sterile onion roots on the surface, mycelia and pycnidia were recovered. Variance and cluster analysis showed similar results among the cultivars with various levels of resistance, tolerance and susceptibility as determined by scoring scales.     

Adzuki bean leaf infection by <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis> and resistance evaluation using detached leaves inoculated with zoospores

We examined the response of adzuki bean leaves to infection by Phytophthora vignae f. sp. adzukicola and determined whether inoculated leaves can be used to evaluate cultivar resistance. Detached adzuki bean leaves were inoculated with zoospores, and the resulting symptoms were diagnosed. Resistant reactions were characterized by dark brown, speckled lesions or a lack of symptoms, while susceptible reactions were characterized by water-soaked spreading lesions. In an inoculation experiment using a combination of three differential cultivars and three races, the response of 10-day-old primary leaves accurately differentiate between race-specific resistance and susceptibility of adzuki cultivars.     

Factors Influencing Transmission of Didymella rabiei (Ascochyta Blight) from Inoculated Seed of Chickpea Under Controlled Conditions

Ascochyta blight of chickpea (Cicer arietinum), caused by the fungus Didymella rabiei, has the potential to cause 100% crop loss in severe epiphytotics. Management of this disease often involves reducing sources of inoculum. The influence of sowing depth, host resistance, seed infection level and soil temperature on disease transmission was investigated in a series of glasshouse and growth room trials using seed artificially inoculated with D. rabiei. A positive correlation (R²=0.9992) was observed between rate of seed infection and the incidence of disease on seedlings. Disease transmission to seedlings was not significantly influenced by sowing depth (1, 3 and 6 cm) in separate trials on two cultivars. Susceptibility of the host showed no obvious influence on the frequency of disease transmission in two trials conducted using four cultivars ranging from highly susceptible to moderately susceptible/moderately resistant. Trials conducted in controlled conditions showed that there was no obvious relationship between soil temperature (5, 9, 14 and 19 °C) and the incidence of disease on seedlings.     

Regulation of defense-related enzymes associated with bacterial spot resistance in Tomato

Twenty tomato (Solanum lycopersicon) cultivars were screened for resistance against bacterial spot disease incited byXanthomonas axonopodis pv.vesicatoria under field conditions with and without pathogen infection. Screening was done by artificially inoculating aX. axonopodis pv.vesicatoria suspension to 4-week-old tomato seedlings and observing them for typical symptoms of the disease. Seedlings were categorized into highly resistant, resistant, susceptible and highly susceptible cultivars on the basis of disease incidence. Tomato cultivars were screened for defense-related enzymes, total phenols and lignin contents. The temporal patterns of all these enzymes were estimated with a moderately susceptible tomato cultivar. Native PAGE analysis of both peroxidase (POX) and polyphenol oxidase (PPO) was carried out for the time course of enzyme activities and also by selecting three different tomato cultivars, following infection with the pathogen. Based on the inducible amounts of these enzymes upon pathogen infection, the tomato cultivars were correlated with the disease incidence under field conditions. A significant (P≤0.05) correlation was observed between the degree of host resistance and the enzyme levels. In highly resistant tomato cultivars the enzyme levels, total phenols and lignin contents were increased in comparison with highly susceptible tomato cultivars. Isoform analysis of POX and PPO enzymes indicated a clear difference between resistant and susceptible tomato cultivars in the number of isoforms and also in the intensity of each isoform in the presence of pathogen infection. The possible regulation of defense-related enzymes in imparting host resistance is discussed. http://www.phytoparasitica.org posting March 11, 2008.     

Emmer wheat, a potential new host of Tilletia indica

Two representative Italian emmer wheat (Triticum dicoccum) landraces, two selected lines and three improved emmer wheat cultivars, derived from crosses with durum wheat (Molisano landrace × ‘Simeto’), were tested for their susceptibility to Tilletia indica, the cause of Karnal bunt of wheat. Plants of emmer wheat were inoculated by injecting allantoid sporidial suspensions into the boot cavity of plants, just prior to ear emergence. A highly susceptible Indian spring wheat cultivar (Triticum aestivum) was used as a comparative control. At maturity of the plants, the seeds were harvested and assessed for incidence and severity of disease. All emmer wheat genotypes tested were infected but showed differing levels of susceptibility. The percentage of infected seeds for individual genotypes ranged from 5.4 to 75.0% compared with 99.1% for WL-711. The severity of infection was less in the old landraces, but it was higher in all the improved emmer wheat cultivars. In conclusion, Italian cultivars of emmer wheat were found to be highly susceptible to T. indica, and are potentially able to support the establishment of the pathogen.Authors L. Riccioni and M. Valvassori contributed equally to this work and should both be considered as first author.     

Prehaustorial Resistance against Alfalfa Rust (Uromyces striatus) in Medicago truncatula

The legume species Medicago truncatula is gaining interests as a plant for structural and functional genomics that can be used to identify agronomically important genes in crop legumes. Resistance to the alfalfa rust (Uromyces striatus) was studied in a germplasm collection of M. truncatula. Accessions varied in resistance, as expressed by disease severity, but none showed macroscopically visible necrosis. Histological investigations, in selected lines covering the whole range of resistance reactions, revealed little difference in spore germination and none in orientation of germtubes on the leaf surface. However, appressorium formation on the stoma was significantly reduced in some accessions. Differences in resistance among accessions were more evident once the stoma were penetrated by the infection structures. Resistance was mainly due to a restriction of haustorium formation with varying levels of early abortion of the colonies, a reduction in the number of haustoria per colony, and hampered colony growth. In addition, necrosis of the host cells associated with infection hyphae was detectable in some accessions from the beginning of colony development. This information will be useful for eventual mapping and cloning analyses of resistance genes in M. truncatula that will in turn be useful for understanding other legume/rust interactions.     

Control of Loose Smut (Ustilago nuda and U. tritici) Infections in Barley and Wheat by Foliar Applications of Systemic Fungicides

Foliar application of a number of broad-spectrum systemic fungicides (particularly conazole fungicides) to loose smut-infected plants of wheat and barley in a 3-spray programme resulted in a marked reduction in the percentage of plants producing infected ears. Such foliar fungicides may have a previously unseen role in reducing the levels of loose smut infection in wheat and barley.     

Differentiation of Bean pod mottle virus isolates based upon host symptoms

Accessions from Glycine, Phaseolus, and Vigna genera were screened for their reactions to different subgroups of isolates of Bean pod mottle virus (BPMV) in order to establish a differential host system. Screening results indicated that the BPMV isolates differed in pathogenic aggressiveness but not in virulence. No major resistance genes were found in soybean (Glycine max) or G. soja since all screened accessions showed mosaic or necrotic symptoms to BPMV inoculation. However, these accessions expressed differences in severity of symptoms when challenged by various BPMV isolates. The inoculation of G. tomentella accessions did not result in mosaic symptoms, and some accessions did not support systemic infection of some of the isolates. Resistance, presented as a hypersensitive reaction, was observed in some of Phaseolus and Vigna genotypes, and resistant response or susceptibility was stable to all the isolates used in the screening. In conclusion, the selected G. soja genotypes PI 407019, PI 464889A, and PI 464928, and ‘Amsoy 71’ soybean may help to separate severe (reassortant) from mild isolates of BPMV based upon their phenotypic reactions.     

Bacterial fruit blotch of melon: screens for disease tolerance and role of seed transmission in pathogenicity

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax avenae subsp. citrulli, is a serious threat to the watermelon and melon industries. To date, there are no commercial cultivars of cucurbit crops resistant to the disease. Here we assessed the level of tolerance to bacterial fruit blotch of various commercial cultivars as well as breeding and wild lines of melon, using seed-transmission assays and seedling-inoculation experiments. Selected cultivars were also tested in a greenhouse experiment with mature plants. All tested cultivars/lines were found to be susceptible to the pathogen, and most of them showed different responses (relative tolerance vs. susceptibility) in the different assays; however, some consistent trends were found: cv. ADIR339 was relatively tolerant in all tested assays, and cv. 6407 and wild lines BLB-B and EAD-B were relatively tolerant in seed-transmission assays. We also provide evidence supporting a strong correlation between the level of susceptibility of a cultivar/line and the ability of the pathogen to adhere to or penetrate the seed. To the best of our knowledge, this is the first attempt to assess melon cultivars/lines for bacterial fruit blotch response.     

Partial stem and leaf resistance against the fungal pathogen <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in wild relatives of tomato

Tomato (Solanum lycopersicum) is one of many greenhouse crops that can be infected by the necrotrophic ascomycete Botrytis cinerea. Commercial cultivation of tomato is hampered by the lack of resistance. Quantitative resistance has been reported in wild tomato relatives, mostly based on leaf assays. We aimed to identify wild tomato relatives with resistance to B. cinerea based on quantitative assays both on leaves and stem segments, monitoring infection frequency and disease expansion rate as parameters. A quantitative tomato stem segment assay was developed. This stem assay and a previously described leaf assay were used to screen a collection of 22 Solanum accessions. Significant differences in disease parameters were observed among accessions. Resistance to B. cinerea was observed in a number of wild Solanum species, including accessions of S. chilense, S. habrochaites and S. neorickii, both in the leaf assay and the stem segment assay. A number of resistant and susceptible accessions were evaluated as adult plants under greenhouse conditions. The data obtained in greenhouse assays confirmed the leaf and stem disease data. The expression of several defence-related genes was studied in a subset of accessions. There was no apparent correlation between the expression levels of the genes tested and the quantitative resistance level to B. cinerea. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Responses of cucumber cultivars to induction of systemic resistance against anthracnose by plant growth promoting fungi

Initial experiment on the reactions of five Japanese cultivars of cucumber toColletotrichum orbiculare infection in the greenhouse revealed that cv Suyo and Gibai were susceptible and moderately susceptible, respectively, while cv Shogoin fushinari and Sagami hanjiro were resistant to infection byC. orbiculare; cv Ochiai fushinari was moderately resistant. The ability of 16 plant growth promoting fungi (some isolates belonged to species ofPhoma and some non-sporulating isolates) isolated from zoysiagrass rhizospheres to induce systemic resistance in the above five cucumber cultivars was tested by growing plants in potting medium infested with barley grain inocula of PGPF in the greenhouse. The second true leaves of 21-day-old plants were challenge inoculated withC. orbiculare and disease assessed. Nine, out of 16 isolates, caused significant reduction of disease caused byC. orbiculare in at least two cultivars.Phoma isolates (GS8-1 and GS8-2) and non-sporulating isolates (GU21-2, GU23-3, and GU24-3) significantly reduced the disease in all the five cultivars. The disease suppression in cucumber was due to the induction of systemic resistance, since the inducer(s) and the pathogen were separated spatially and that the inducer did not colonize aerial portions. The resistance induced by certain isolates in a susceptible cultivar was less than that in a resistant cultivar. Disease suppression caused by isolate GU21-2 was similar to theC. orbiculare induced control in certain cultivars. The average rate of expansion of lesion diameter on leaves due toC. orbiculare was slower due to induction with the selected plant growth promoting fungi compared to the uninduced control plants. Roots of four cultivars were colonized by only three isolates, however, roots of one cultivar (Suyo) was colonized by five isolates suggesting the cultivar-specific root colonization ability.Abbreviations cv cultivar(s) - PGPF plant growth promoting fungal isolates - PGPR plant growth promoting rhizobacteria