<Emphasis Type="BoldItalic">Didymella glomerata</Emphasis><Emphasis Type="Bold">causing leaf blight on pistachio</Emphasis>

During 2015 and 2016, we detected blighted leaves of pistachio (Pistacia vera) trees in different orchards in Arizona (USA). A Phoma-like species was isolated from pycnidia that appeared embedded in the leaf tissue. The pathogen was identified by means of morphological characteristics and DNA analysis (by sequencing of the ITS, BT, and EF regions) as Didymella glomerata. The optimum temperature for mycelial growth of the pathogen was around 25 °C. Inoculation tests were conducted on healthy and wounded pistachio leaves, fruits, and shoots of the cv. Kerman (female) and the cv. Peters (male). Overall, the pathogen was highly virulent on leaves of both pistachio cultivars and did not need injuries for infection. Conversely, the pathogen did not cause any macroscopic symptoms on the inoculated fruits and shoots but showed a certain endophytic behavior in the shoots. Also, data on the ability of different fungicides to inhibit the in vitro mycelial growth of the pathogen are presented.     

Comparative biology and life tables of <Emphasis Type="BoldItalic">Trichogramma aurosum</Emphasis> on <Emphasis Type="BoldItalic">Cydia pomonella</Emphasis> at constant temperatures

The influence of constant temperatures on biological parameters of German strains of Trichogramma aurosum Sugonjaev & Sorokina (Hymenoptera: Trichogrammatidae) was evaluated in the laboratory on eggs of Cydia pomonella L. (Lepidoptera: Tortricidae). Development time and longevity of all strains were decreased as temperature increased. Development time of the strains differed significantly only when exposed to 15°, 20°, and 25°C. Cumulative fertility and longevity differed significantly at 15° and 20°C. Realized fertility differed significantly at all constant temperatures. Emergence rates of all strains were less than 65% and were decreased even further as temperature increased. Female-biased sex ratio ranged from 65% to 100% at all constant temperatures. The low temperature threshold for T. aurosum was 10°C and the mean number of degree-days at 15°, 20°, 25° and 30°C was 175, 183, 173 and 185, respectively. The Bavarian strain tolerated high temperatures and had the highest parasitization capability, while the Hessian strain had the lowest parasitization at all temperatures. Fertility life table analysis revealed a major effect of temperature on the population growth parameters. Net reproductive rate was highest at intermediate constant temperatures in all strains, with the highest rate recorded for the Bavarian strains at all constant temperatures. Mean cohort generation time, and population doubling time decreased as temperature increased. The daily intrinsic rate of increase and finite rate of increase were positively correlated with temperature. The relevance of our results is discussed in the context of climatic adaptation, intraspecific variability and biological control.     

Calcium fertilizers induce soil suppressiveness to <Emphasis Type="BoldItalic">Phytophthora cinnamomi</Emphasis> root rot of <Emphasis Type="BoldItalic">Quercus ilex</Emphasis>

Based on the observation that the root disease caused by P. cinnamomi on Q. ilex has a low incidence and severity in soils with medium-high Ca²⁺ content, we studied the ability of Ca²⁺ fertilizers to induce soil suppressiveness to the pathogen. Studies on cultures of P. cinnamomi exposed to different Ca²⁺ fertilizers in vitro showed significant inhibition of sporangial, chlamydospore and zoospore production at millimolar concentrations while mycelial growth was mainly unaffected. Experiments performed with artificially infested soil showed that some Ca²⁺ fertilizers induce a significant decrease on chlamydospore viability. Additionally, greenhouse experiments using artificially infested soils showed a significant reduction of foliar and root symptom severities in Holm oak seedlings growing in soils amended with Ca²⁺ fertilizers. We suggest that limestone amendments in oak rangelands could enhance the suppressiveness of soils to P. cinnamomi, and it is likely that the inhibition of sporangial production was the main mechanism involved.     

Insect growth-regulating effects of <Emphasis Type="BoldItalic">Withania somnifera</Emphasis> in a polyphagous pest, <Emphasis Type="BoldItalic">Spodoptera litura</Emphasis>

Treatment of sixth instar larvae and pupae of the polyphagous pest Spodoptera litura Fabr. (Lepidoptera: Noctuidae) with an acetone extract of leaves of Ashwagandha, Withania somnifera L. (Solanaceae), caused toxicity, molt disturbances, formation of larval–pupal, pupal–adult intermediates and adultoids. Our results suggest that W. somnifera acts as an insect growth regulator causing disruption of the endocrine mechanism regulating molting and metamorphosis.     

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.     

<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.     

Assessing genetic resistance to spot blotch, <Emphasis Type="Italic">Stagonospora nodorum</Emphasis> blotch and tan spot in wheat from Nepal

Fungal leaf spot diseases of wheat (Triticum aestivum L.) in Nepal cause significant yield reduction. Although field testing has identified a few partially resistant cultivars, most wheat grown in Nepal lacks adequate resistance to leaf spot diseases. During 2009–2010, 116 local and commercial spring wheat cultivars and advanced breeding lines were selected from multi-year field experiments in Nepal and evaluated for seedling resistance to three leaf spot diseases: spot blotch, Stagonospora nodorum blotch (SNB) and tan spot races 1 and 5 (two of the most prevalent races) in the growth chambers at North Dakota State University, Fargo, ND, USA. The wheat cultivars and lines were artificially inoculated with individual pathogens or races at the two-leaf stage and disease reactions were evaluated 6 to 10 days after inoculation (DAI). Results indicated that 30%, 31%, 19% and 10% of the tested wheat cultivars and lines were resistant to spot blotch, SNB, tan spot races 1 and 5, respectively. Six advanced breeding lines (SW89-5422, BL 2127 = DANIAL88/HLB30//NL297, BL 3033, FILIN/IRENA/5/CNDO/R143//ENTE/MEXI-2/3/AE. SQUA (TAUS)/4WEAVER, GAN/AE.SQUARROSA (236)//DOY1/AE.SQUARROSA(447)/3/MAIZ/4/INQALAB91, Mayoor//TK SN1081/Ae. Squarrosa (222)/3/FCT, were resistant to spot blotch, SNB and tan spot race 1. Similarly, two wheat cultivars Chirya 3 and Chirya 7 were resistant to spot blotch, and tan spot races 1 and 5. The resistant wheat lines identified in this study represent potentially useful and untapped sources of resistance to multiple leaf spot diseases and should be utilized in wheat breeding programs in Nepal in order to develop wheat cultivars with broad-spectrum resistance.     

Evaluation of the <Emphasis Type="BoldItalic">Sss</Emphasis> AgriStrip rapid diagnostic test for the detection of <Emphasis Type="BoldItalic">Spongospora subterranea</Emphasis> on potato tubers

Spongospora subterranea, f.sp. subterranea (Sss), which causes powdery scab, is mainly spread through infected seed tubers and survives in contaminated soil for many years. The visual assessment of tuber lots by inspectors carries the risk of misidentification due to the difficulty of distinguishing lesions caused by either Sss or Streptomyces spp.. To avoid this, the “Sss AgriStrip”, a rapid and lab-independent test tool based on a lateral flow immunoassay has been developed, and we assessed its accuracy and sensitivity for detecting Sss. The Sss AgriStrip performed as well as other lab-based identification methods. The Sss AgriStrip, microscopy, ELISA, PCR, and real-time PCR techniques identified infection with S. subterranea in all tubers with typical powdery scab lesions. When lots with tubers showing a mixture of typical and atypical (suspicious) symptoms were tested, the presence of S. subterranea was confirmed in all lesions by all methods. The DNA content was generally lower in atypical than in typical lesions. Diverse and suspicious symptoms, which were difficult to assign to either powdery or common scab, tested negative with Sss AgriStrip and the other methods. This was despite microscopic observation of sporosori-like structures in some samples. Isolation and molecular identification confirmed that these lesions were mostly caused by Streptomyces spp. The Sss AgriStrip is as sensitive as DAS-ELISA with a detection limit between 1 and 10 sporosori per ml buffer. It is ideal for rapid and selective detection of Sss on farms and border inspection points to prevent spread of the pathogen.     

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.     

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.     

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.     

Bacterial black spot caused by <Emphasis Type="Italic">Burkholderia andropogonis</Emphasis> on <Emphasis Type="Italic">Odontoglossum</Emphasis> and intergeneric hybrid orchids

A new bacterial black spot disease was observed on Odontoglossum, Odontioda, Odontocidium, and Vuylstekeara orchids in Japan. Typical symptoms on the leaves were dark or black spots (or both) with a yellow halo. The causal agent was identified as Burkholderia andropogonis (Smith 1911) Gillis, Van Van, Bardin, Goor, Hebbar, Willems, Segers, Kersters, Heulin and Fernandez 1995. The isolates were pathogenic on four original host orchids, Phalaenopsis orchid, and tulip; they were not pathogenic on white clover or corn after needle stab inoculation. An antibiotic bactericide (oxytetracycline/streptomycin mixture WP) was most effective for controlling the disease.     

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.     

Encapsulation rates of the parasitoid <Emphasis Type="BoldItalic">Anagyrus</Emphasis> sp. nr. <Emphasis Type="BoldItalic">pseudococci</Emphasis>, by three mealybug species (Hemiptera: Pseudococcidae)

Laboratory studies assessing the degree of suitability of the mealybugs Planococcus ficus (Signoret), Planococcus citri (Risso) and Pseudococcus calceolariae (Maskell), towards the Sicilian ecotype of the encyrtid parasitoid Anagyrus sp. nr. pseudococci were carried out. All three species of mealybugs were shown to encapsulate the eggs of the encyrtid; however, significant differences were revealed among them in rates of encapsulation and/or superparasitism. The level of aggregate encapsulation and effective encapsulation of the parasitoid eggs by P. citri (74.95 ± 0.87 and 60.19 ± 1.70, respectively) was significantly higher than that recorded for P. ficus (aggregate encapsulation = 58.43 ± 0.83 and effective encapsulation = 31.31 ± 1.55) and significantly lower than the values found in Ps. calceolariae (93.99 ± 0.97 and 88.61 ± 2.03, respectively). As such, rates of parasitism were significantly lower for P. ficus compared with both P. citri and Ps. calceolariae. The two latter species of mealybugs showed similar rates of parasitism by A. sp. nr. pseudococci. Moreover, a significantly higher rate of superparasitism was found for Ps. calceolariae compared with both P. citri and P. ficus, whereas no significant differences were found between the two species of Planococcus for this parameter.     

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.     

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.     

Natural phenolic acids from wheat bran inhibit <Emphasis Type="Italic">Fusarium culmorum</Emphasis> trichothecene biosynthesis <Emphasis Type="Italic">in vitro</Emphasis> by repressing <Emphasis Type="Italic">Tri</Emphasis> gene expression

The effect of natural phenolic acids from wheat bran on type B trichothecene biosynthesis by Fusarium culmorum was investigated in vitro. Durum wheat bran contained various monomeric forms of phenolic acids, with ferulic acid being the most abundant. In addition, various oligomeric forms of ferulic acid and mainly dimeric forms were also detected. When liquid cultures of F. culmorum were supplemented with a natural wheat bran extract, trichothecene production was fully inhibited. The exact mechanism by which toxin synthesis is repressed remains to be clarified but we showed that the phenolic acid treatment resulted in a drastic reduction in the expression level of structural trichothecene biosynthetic genes. The inhibitory efficiency of the natural phenolic acid extract was significantly higher than that of a reconstituted mixture containing similar concentrations of monomeric forms. Thus, to elucidate the full repression of type B trichothecene production induced by the natural phenolic acid extract from wheat bran, two hypotheses can be raised: (i) a synergistic impact of monomeric and dimeric forms of phenolic acids, (ii) the occurrence of an unidentified oligomeric form able to efficiently repress toxin yield. As a first attempt to investigate the effect of oligomeric forms, one of the most abundant dimer of ferulic acid, the 8-5′-benzofuran dimer, has been synthesized in vitro and was shown to inhibit trichothecene biosynthesis to the same extent than the monomer of ferulic acid.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

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.