Survey of mealybug (Hemiptera: Pseudococcidae) vectors of <Emphasis Type="Italic">Ampelovirus</Emphasis> and <Emphasis Type="Italic">Vitivirus</Emphasis> in vineyards of northwestern Italy

Mealybugs (Hemiptera: Pseudococcidae) are sap-sucking insects which infest a broad range of crops worldwide. They represent an important threat to viticulture as they are vectors of viruses associated with leafroll and rugose wood complex diseases. In this study, we surveyed the presence of mealybugs and their associated viruses in vineyards of the Piemonte and Liguria regions, northwestern Italy. In order to determine the collected specimens correctly, we added a species-specific marker for Heliococcus bohemicus to an existing molecular identification key. The only species collected in Piemonte was H. bohemicus, whereas in Liguria, H. bohemicus, Planococcus ficus and Pseudococcus longispinus were found; Ps. longispinus has never before been reported in Italian vineyards. Several specimens of all three species were infected by the ampeloviruses GLRaV-1 and GLRaV-3 and the vitivirus GVA. Both nymphs and adult females tested positive for the viruses and mixed infections were commonly found within the same insect. Population levels and virus incidence were higher in Liguria than in Piemonte, suggesting a greater risk of disease spread. We conclude that the mild, Mediterranean climate of Liguria favors the development of a diverse mealybug fauna while only H. bohemicus, known to be tolerant to the severe continental winter temperatures, colonize grapevines in colder viticultural areas.     

Data on the ecology of <Emphasis Type="Italic">Kermes echinatus</Emphasis> Balachowsky (Hemiptera,Coccomorpha, Kermesidae) on <Emphasis Type="Italic">Quercus ilex</Emphasis> (Fagaceae) in Greece

The Phenology and natural enemies of the scale insect Kermes echinatus Balachowsky (Hemiptera: Kermesidae), which infests the holly oak Quercus ilex (Fagaceae), were studied between November 2015 and November 2017, in southern Greece (Kalamata, Peloponnese). Kermes echinatus is a univoltine, oviparous and biparental species. It overwinters as 1st instar nymphs, settling on the branches of the trees. The second instar nymphs appear by the middle of April, followed by 3rd instar nymphs until the end of May. The male larvae and pupae were recorded from the end of April to the end of May. Mature females, pre-ovipositing and ovipositing adults, were recorded during May and June. The newly hatched crawlers appeared by the end of June, which remained at this stage, settled on branches during the rest of summer and winter periods till next April. The parasitoid species Metaphycus gennaroi Guerrieri & Noyes and the hyperparasite Cheiloneurus claviger Thomson (Hymenoptera: Encyrtidae) were recorded on the colonies of the scale. The parasitism level of the scale reached to 21%. The predator Chilocorus bipustulatus L. (Coleoptera: Coccinellidae) was also recorded on the colonies of the scale.     

Parasitoid complex of <Emphasis Type="Italic">Planococcus ficus</Emphasis> (Signoret) (Hemiptera: Pseudococcidae) in Turkey

In this study a survey for determining the parasitoid complex of Planococcus ficus (Signoret) (Hemiptera: Coccomorpha: Pseudococcidae) was conducted in Adana and Mersin Provinces, Turkey. Five primary, two primary/secondary, two secondary parasitoid species associated with P. ficus, were found. Clausenia josefi Rosen and Chartocerus kurdjumovi (Nikol’skaya) are new records for Turkish fauna.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

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.     

Anthracnose of <Emphasis Type="Italic">Enkianthus campanulatus</Emphasis> and <Emphasis Type="Italic">Rhynchosia acuminatifolia</Emphasis> caused by <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> (new occurrence)

In 2003–2004, anthracnoses of Enkianthus campanulatus and Rhynchosia acuminatifolia were found for the first time in Kanagawa Prefecture and Tokyo in Japan. These pathogens were identified as Colletotrichum gloeosporioides based on their pathogenicity, morphology and ribosomal DNA spacer sequences. Results were presented at the annual meeting of The Phytopathological Society of Japan in 2004.     

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.     

Natural enemies of <Emphasis Type="Italic">Diaspis echinocacti</Emphasis> in Greece and first records of <Emphasis Type="Italic">Aphytis debachi</Emphasis> and <Emphasis Type="Italic">Plagiomerus diaspidis</Emphasis>

Two hymenopteran parasitoids of the cactus scale Diaspis echinocacti (Bouché) (Hemiptera: Diaspididae) on Opuntia ficus-indica (L.) Mill. (Cactaceae) are recorded in Greece. Aphytis debachi Azim, 1963 (Aphelinidae) is first recorded for Europe and Plagiomerus diaspidis Crawford, 1910 (Encyrtidae) is first recorded for Greece. Preliminary data on phenology and natural enemies of the scale D. echinocacti on O. ficus-indica are presented. Parasitism of D. echinocacti by P. diaspidis reached 86% in southern Greece (Kalamata) and parasitism by A. debachi reached 9.3% and 12% in Kalamata and Athens, respectively. Two predators, Cybocephalus fodori Endrödy-Youga (Coleoptera: Nitidulidae) and a mite species (Prostigmata: Bdellidae), were found to be associated with D. echinocacti.     

<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.     

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.     

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.     

Effects of pH and titratable acidity on the growth and development of <Emphasis Type="Italic">Monilinia laxa</Emphasis> (Aderh. &amp; Ruhl.) <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>.

This investigation examines the effects of pH and titratable acidity on the growth and developments of a strain of Monilinia laxa (Aderhold & Ruhland) at seven different pH levels in Potato Dextrose Agar media and on peach fruit from formation to commercial maturity. The fungi growth was obtained by daily measurement of mycelia on the pH amended Potato Dextrose Agar. The sporulation performance was determined after 30 days of culture incubation. Fruits were inoculated with M. laxa, from fruit set to maturity, on weekly basis for brown rot susceptibility. The pathogen development, in vitro, was affected, by the pH (2.4–11.52) amended nutrient media. M. laxa exhibited variation in its growth and sporulation capacities on the seven pH amended PDA, preferring relatively moderate acidic conditions for optimum performance. In the in vitro analysis, there was mycelia growth at pH 2.40 to 8.84, while pH 11.52 did not support any mycelia growth. There was a continuous and stable increase in weight of fruit as it developed whereas the fruit size increased, then decreased and finally increased as the fruit develops. The acidity dynamics exhibited a non-sinusoidal waveform through the growth and development of the fruit. In all these characteristic variations, M. laxa did not develop infection or shown any brown rot incidence in the fruit until the period of commercial maturity.     

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.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

<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.     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

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.     

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.