Gene-based analysis of <Emphasis Type="Italic">Puccinia</Emphasis> species and development of PCR-based marker to detect <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> causing yellow rust of wheat

Stripe rust is considered as the current major rust disease affecting winter cereal production across the world. A quick, reliable PCR-based marker was developed here to detect, identify and rapidly monitor Puccinia striiformis f. sp. tritici (Pst) in wheat-growing areas. Three respective sets of primers, designed from β-tubulin, squalene monooxygenase and ketopantoate reductase genes selected from the full genome of Puccinia striiformis f. sp. tritici, amplified sequences of 239, 358 and 1518 bp, respectively, in Pst pathotypes. A fragment of 1518 bp unique to Pst pathotypes was amplified using primer set PstKeto F1_30/Pst KetoR1_1547 and distinguished the pathogen clearly from different Puccinia spp. and other fungal pathogens. The detection limit of the marker (KetoPstRA₁₅₀₀, accession no. KU240073) by conventional PCR assay was 10 pg. This marker could detect the pathogen in the host before symptom expression. The sensitivity and utility of the marker were further enhanced in a qPCR-based assay that was developed with a newly designed primer set PstKeto F1_1246/Pst KetoR1_1547, which amplified a product of 302 bp and detected as little as 10 fg of DNA. This PCR/qPCR based marker is suitable for studying cultivar resistance, which requires accurate quantification of the pathogen in diseased host tissue.     

Identification and characterization of pathotypes in <Emphasis Type="Italic">Puccinia horiana</Emphasis>, a rust pathogen of <Emphasis Type="Italic">Chrysanthemum</Emphasis> x <Emphasis Type="Italic">morifolium</Emphasis>

Puccinia horiana is the causal agent of chrysanthemum white rust or Japanese rust. This microcyclic autoecious rust has a quarantine status and can cause major damage in the commercial production of Chrysanthemum x morifolium. Given the international and often trans-continental production of planting material and cut flowers of chrysanthemum and the decreasing availability of registered fungicides in specific regions, breeding for resistance against P. horiana will gain importance and will need to involve the appropriate resistance genes for the pathotypes that may be present. As pathotypes have not been well characterized in this system, the main objective was to build an international collection of isolates and screen these on a large collection of cultivars to identify different pathotypes. Using a robust and high throughput bioassay, we tested 36 selected cultivars with 22 individual single-pustule isolates of P. horiana. The isolates originated from three different continents over 4 different collection years and included some isolates from cultivars previously reported as resistant. In most cases the bioassays resulted in a clear scoring of interaction phenotypes as susceptible or resistant, while in several cases consistent intermediate phenotypes were found, often on specific cultivars. Twenty-four of the cultivars gave a differential interaction phenotype profile. All isolates produced a unique profile, infecting a minimum of 4 and a maximum of 19 differential cultivars. Based on the Person analysis of these profiles, this pathosystem contains at least seven resistance genes (and seven avirulence genes), demonstrating the highly complex race structure in this pathosystem.     

Effect of controlled fluctuating low temperatures on survival of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important disease of wheat worldwide. Understanding the survival of Pst during the winter is critical for predicting Pst epidemics in the spring. We used a real-time quantitative PCR (qPCR) method to quantify Pst CYR32 biomass in infected wheat seedlings under several fluctuating temperature regimes (three average temperatures 0, ?5 and ?10 °C, each with two daily fluctuating amplitudes 8 and 13 °C). The survival of Pst CYR32 increased with increasing average temperature but also varied greatly with the amplitude – larger amplitude led to lower survival, particularly at 0 and ?5 °C. Nevertheless the survival at both amplitudes was still significantly greater than under the corresponding constant temperatures. There were small, albeit statistically significant, differences between the two cultivars (Xiaoyan 22, low winter-hardiness; Lantian 15, high winter-hardiness) in Pst CYR32 survival. This study indicated potential errors that could result from using daily average temperatures to predict Pst survival during the winter.     

Spatial genetic diversity and interregional spread of <Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis> in Northwest China

In China, wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat. The Longnan and Linxia regions in Gansu Province and Qinghai Province are the major over-summering regions for the pathogen and key epidemiological zones in Northwest China. Population genetic diversity and interregional long-distance spread of the wheat stripe rust pathogen in Northwest China were studied using SSR markers. The genetic diversity in the Longnan population was much higher than those in the Linxia and Qinghai populations. Therefore, the molecular data confirmed that the Longnan region is a center of genetic diversity for P. striiformis f. sp. tritici in Northwest China. The low genetic differentiation (Gst = 0.15) and the extensive gene flow (Nm = 1.37) were found among the three regions in Northwest China. The most important conclusion of this study is that the stripe rust inoculum in Qinghai can come from both Longnan and Linxia, but mainly from Longnan directly in the spring.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

The development of a PCR-based method for detecting <Emphasis Type="Italic">Puccinia striiformis</Emphasis> latent infections in wheat leaves

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici is one of the most important diseases on wheat worldwide, especially in temperate regions with cool moist weather conditions. A rapid and reliable detection of the pathogen in latent infected wheat leaves during overwintering of the fungus in the dormant stage will contribute to determine the initial inoculum potential and thus to predict early outbreak and to improve effective management of the disease. To achieve this aim, a PCR-based method was developed for specific and sensitive detection of P. striiformis. Specific primers were designed according to a genome-specific sequence of P. striiformis. To evaluate the specificity of the primers, seven different isolates and races of P. striiformis as well as six other pathogens of wheat were tested. All isolates of P. striiformis yielded a distinct band of a fragment of 470 bp, while using DNA of the other wheat pathogens as a template no amplification product was detected. The sensitivity of the primers was tested using serial dilutions of total DNA from P. striiformis; the limit of detection was 10 pg of DNA. Using extracts from P. striiformis-infected wheat leaves, the fungus could be determined in the leaves before symptoms appeared. The stripe rust could also be detected in the dormant stage by the PCR assay in samples of wheat leaves taken during the winter season. The application of the PCR assay may be useful for rapid and reliable detection of P. striiformis in latent infected leaves of overwintering wheat plants.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

Resistance to <Emphasis Type="Italic">Penicillium</Emphasis><Emphasis Type="Italic">hirsutum</Emphasis> Dierckx in garlic accessions

Among the factors affecting the quality and yield of garlic production, blue mold caused by -- Penicillium spp. -- is responsible for economical losses in many countries. Allicin, present in garlic bulbs, has been suggested as having antifungal activity against some Penicillium species. This study was conducted to evaluate the response of garlic accessions against Penicillium hirsutum infection and to compare this response with bulb allicin content. Twelve garlic accessions were inoculated with P. hirsutum, and assayed in greenhouse and growth chamber experiments. Plant growth parameters and the fungal production of conidia were evaluated. Significant differences were found among the accessions. Accessions Castaño and Morado were most resistant whereas AR-I-125 and Fuego were always severely affected by the disease. A low correlation was found (r = 0.17) between allicin content and tolerance, indicating that allicin is not the main factor involved in the resistance against P. hirsutum.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.     

White rust of <Emphasis Type="Italic">Ipomoea</Emphasis> caused by <Emphasis Type="Italic">Albugo ipomoeae-panduratae</Emphasis> and <Emphasis Type="Italic">A. ipomoeae-hardwickii</Emphasis> and their host specificity

In some areas of Japan, yellow spots with white pustules on leaves, stems, petioles, peduncles and calyces were found on Ipomoea nil, I. triloba, I. lacunosa and I. hederacea var. integriuscula. We demonstrated that the diseases on I. nil, I. triloba and I. lacunosa were caused by host-specific strains of Albugo ipomoeae-panduratae and defined three forma speciales of the fungus, respectively, for the three Ipomoea species: “f. sp. nile”, “f. sp. trilobae” and “f. sp. lacunosae”. Because the diseases were new to Japan, we coined the Japanese name “shirosabi-byo”, which means white rust. We also showed that the disease on I. hederacea var. integriuscula was caused by A. ipomoeae-hardwickii. We named this new disease “white rust (shirosabi-byo in Japanese)”.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

<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.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Effect of seed priming with <Emphasis Type="Italic">Serratia plymuthica</Emphasis> and <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> to control <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in different oilseed rape cultivars

The efficacy of a seed treatment of oilseed rape (OSR) (Brassica napus) with the rhizobacteria Serratia plymuthica (strain HRO-C48) and Pseudomonas chlororaphis (strain MA 342) applied alone or in combination against the blackleg disease caused by Leptosphaeria maculans was tested with different cultivars. Seeds were soaked in bacterial suspensions (bio-priming) to obtain log₁₀ 6–7 CFU seed⁻¹. Cotyledons were inoculated with a 10 ul droplet of L. maculans spore suspension of log₁₀ 7 spores ml⁻¹ and the disease index (size of lesions) was evaluated 14 days later. A mean disease reduction of 71.6% was recorded for S. plymuthica and of 54% for P. chlororaphis. The combined treatment was not superior to the treatment with S. plymuthica alone. The reduction of the disease caused by S. plymuthica was independent of the cultivar’s susceptibility, whereas the control effect recorded with P. chlororaphis increased with decreasing cultivar resistance to blackleg disease. The bacterial colonization of OSR was restricted to the roots and hypocotyl. No significant difference in bacterial colonization of the rhizosphere was observed between different cultivars, nor between single or combined bacterial seed treatments.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.     

First report of leaf spot caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> on <Emphasis Type="Italic">Drimia maritima</Emphasis>

Drimia maritima (squill) is a historically important medicinal plant. During the spring of 2016, small, yellow leaf spots, which became brown and finally necrotic, were observed on squill plants in Kohgiluyeh and Boyer-Ahmad Provinces in Iran. A fungus was consistently isolated from infected leaves and identified as Alternaria alternata based on morphological and phylogenetic analyses. Pathogenicity tests confirmed A. alternata to be the causal agent of the newly observed leaf spot disease. This is the first report of leaf spot on D. maritima caused by A. alternata in the world.     

Immunodetection of the replicative complex of <Emphasis Type="Italic">Barley yellow dwarf</Emphasis><Emphasis Type="Italic">virus</Emphasis>-PAV <Emphasis Type="Italic">in vivo</Emphasis>

The genomic fragments of two open reading frames (ORFs) 1 and 2 of German and Canadian PAV isolates of Barley yellow dwarf virus (BYDV-PAV) were sequenced. Sequences only slightly differed from previously published sequences of this virus. Two polyclonal antisera against proteins encoded by ORFs 1 and 2 of a German ASL-1 isolate were developed using recombinant antigens expressed in E. coli as a fusion either to His₆− or thioredoxin-tags. In Western blot analysis with total protein extracts from BYDV infected plants, antisera efficiently recognized the 99 kDa fusion protein expressed from ORF1 and ORF2 (P1–P2 protein). Later in infection the P1–P2 protein disappeared and two smaller proteins, revealing sizes of 39 and 60 kDa, could be detected.