Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.     

Presence of less-preferred hosts of the aphid parasitoids <Emphasis Type="Italic">Aphidius ervi</Emphasis> and <Emphasis Type="Italic">Praon volucre</Emphasis> reduces parasitism efficiency

Parasitoids are characterized by a defined range of hosts, either more specialist or generalist. Under natural conditions, females may encounter different host species on the same plant or in the same location. In this case, their preference for one host could influence their choice. However, the presence of less suitable hosts may also affect their choice and, in some cases, may reduce their interest in a patch where both preferred and less preferred hosts are available. The aim of the present study was to test the consequences of the simultaneous presence of three cereal aphids (Sitobion avenae Fabricius, Metopolophium dirhodum Walker, and Rhopalosiphum padi Linnaeus) on the parasitism by two of their parasitoids, Aphidius ervi Haliday and Praon volucre Haliday. Firstly, in the no-choice experiment, A. ervi parasitized on S. avenae at a significantly higher rate as compared to M. dirhodum, whereas no parasitism on R. padi was observed. P. volucre parasitized the three species of cereal aphids with a significant preference for S. avenae. Interestingly, when two or three host species were offered simultaneously in the same quantity to pairs of parasitoids, the level of parasitism was less than that observed for one host species alone. This observation exhibits a distractive effect on non-host species, from the defense mechanism of a non-suitable host or from the perception of bad quality patches. These results raise the question of the practical application of inundative release of parasitoids for biocontrol when several hosts are available simultaneously.     

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.     

Competence of <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> from Cruciferous Weeds and Wallflower (<Emphasis Type="Italic">Erysimum cheiri</Emphasis>) to Induce Black Rot in Cabbage

Aphanomyces euteiches Drechsler is an oomycete pathogen of leguminous crops that causes root rot, a severe disease of pea (Pisum sativum L.) worldwide. An improved understanding of the genetic structure of A. euteiches populations would increase knowledge of pathogen evolution and assist in the design of strategies to develop pea cultivars and germplasm with stable disease resistance. Twenty six primers pairs were used to amplify Sequence Related Amplified Polymorphisms (SRAP) among 49 A. euteiches isolates sampled from pea. A total of 190 polymorphic SRAP bands were generated, of which 82 were polymorphic between all the A. euteiches isolates. The percentage of polymorphic bands per primer pair ranged from 22 to 75%. According to the PIC value estimated for each marker, 60% of the SRAP markers were highly to reasonably informative (PIC > 0.25). Genetic structure of A. euteiches populations sampled in different American and French locations showed low to high genetic diversity within populations. The largest variation occurred within countries, with a total estimated genetic diversity of 0.477 and 0.172 for American and French populations, respectively. This was particularly evident from a principal component analysis (PCA) and a Minimum Spanning Networks (MSN) based on genetic profiles of isolates, which generated two different clusters, one corresponding to the French isolates and four American isolates (MV1, MV5, MV7, Ath3), and the other to American isolates. A. euteiches populations from cultivated pea in France appeared as a single unstructured population, whereas American isolates of A. euteiches diverged into three different populations.     

Molecular diversity in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> isolates from common bean fields in Brazil

The common bean (Phaseolus vulgaris L.) is widely cultivated in Brazil and is known as a very important crop for families in this country. Fusarium wilt severely harms common beans and has become a big issue for this crop. In order to assist the breeding programs that target resistance to this disease, the evaluation of genetic diversity of the pathogen and its molecular characterization are crucial. Thus, the present goal was to identify Fusarium isolates obtained from several places in Brazil using molecular tools; select molecular markers for these isolates; and analyze their diversity. All of isolates were molecularly identified as Fusarium oxysporum f. sp. phaseoli (Fop). By using seven selected SSR markers, the results of diversity obtained by the dendrogram and the Bayesian analysis formed four groups where a large diversity of this fungus was found within each state. However, the groups were more homogenous according to the collection source and the pathogenicity test. More specifically, group 2 was composed of the most virulent strains and originated from Minas Gerais State – UFV, and group 3 was mostly composed by isolates from Goias state. Group I was also more diverse in terms of location and virulence. The overall results indicated a positive correlation between Fusarium diversity and its virulence to common bean. Furthermore, the use of these markers was effective in molecular identification and in detecting polymorphism within F. oxysporum f. sp. phaseoli.     

Population differentiation of <Emphasis Type="Italic">Puccinia coronata</Emphasis> between hosts –implications for the epidemiology of oat crown rust

The fungus Puccinia coronata Corda. is the causal agent of crown rust on oats (Avena sativa) and grasses and the disease is a major problem in oat production causing devastating yield losses. The population biology of P. coronata in oat fields and on the aecial host in central Sweden was studied to get a deeper understanding of the role of the aecial hosts in the epidemiology of the disease. Samples were collected from the aecial hosts common buckthorn (Rhamnus cathartica) and alder buckthorn (Frangula alnus), and three adjacent spring oat (Avena sativa) fields. Microsatellite markers were used to evaluate the relationships between populations sampled from the different hosts. According to our results F. alnus can be excluded as a part of the oat crown rust disease cycle. The results further show that samples collected from the aecial host were genetically separate from the population sampled in adjacent oat fields. Concurrently, the genotypic variation of P. coronata observed within oat fields was high. No population differentiation was observed within or between samples collected from different fields within the region, suggesting that airborne spores from other than the sampled specimens of the aecial hosts were contributing to the genetic diversity of P. coronata f. sp. avenae in the selected oat fields.     

Population genetic structure of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG1IA from rice field in North India

Rhizoctonia solani AG1IA is an important fungal pathogen causing significant yield and quality losses in rice production. However, little is known about the levels of genetic diversity and structure of this pathogen in North India. Out of 240 samples collected from different rice-growing regions of North India, 112 isolates were identified as R. solani AG1IA subgroups using species-specific primers. All 112 isolates were organized into four groups on the basis of percent disease index (PDI). The majority of the isolates were weakly virulent. Population genetic analysis was performed within and between populations using inter simple sequence repeat (ISSR) markers. A total of 8249 alleles were identified from the 112 isolates of R. solani AG1IA through analysis of the ten inter simple sequence repeat markers. All the ten ISSR markers were polymorphic. The average number of bands per primer was 7.3 which ranged in size from 250 to 1500 bp. Genetic structure of the isolates using inter simple sequence repeat primers showed high degree of polymorphism (PIC ≥0.81). The analysis of molecular variance (AMOVA) indicated that most of the genetic diversity occurred within populations (60%), while the variability among populations and among regions contributed 25 and 15%, respectively. Overall, the present study reveals that a large variation exists among rice-infecting isolates of R. solani AG1IA in North India. Fingerprinting of the isolates using ISSRs along with phenotypic characterization and virulence analysis will help epidemiological studies that can provide new insights into pathogen biology and disease spread.     

Host-suitability of black medick (<Emphasis Type="Italic">Medicago lupulina</Emphasis> L.) and additional molecular markers for identification of the pea cyst nematode <Emphasis Type="Italic">Heterodera goettingiana</Emphasis>

A survey was conducted in 16 fields cultivated with broad bean (Vicia faba L.) and garden pea (Pisum sativum L.) in nine localities of Apulia, southern Italy, to determine whether annual weeds were susceptible to the pea cyst nematode (PEACN), Heterodera goettingiana, and could therefore serve as alternate host for the nematode. The results of this study showed that black medick (Medicago lupulina L.) is a good host for the nematode increasing its population levels in the soil in the absence of the primary hosts. The identity of the PEACN was confirmed by integrative taxonomic approaches (classical, and molecular), resulting identical in all cases (broad bean and garden pea, as well as the spontaneous black medick infections). The phylogenetic analyses using ITS and coxI gene regions strongly support the identification of the populations of H. goettingiana from Italy. Also, ITS and coxI gene sequences were obtained from the same cyst, confirming the species identity in comparison to other nematodes and populations in the Goettingiana group, demonstrating that ITS and coxI gene regions of the PEACN are suitable molecular markers for accurate and unequivocal identification of the PEACN. Reproduction and histopathological analyses demonstrated a good host-suitability of black medick to the PEACN. This record enlarges the relatively narrow host-range of the pea cyst nematode and indicates the need to control M. lupulina to avoid the increase of the nematode population in the absence of the main host crop.     

Development of simple sequence repeat markers for the classification of the clubroot pathogen <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in cruciferous crops. To classify isolates, we developed simple sequence repeat (SSR) markers for P. brassicae. Twenty-four Japanese isolates were used in this study: 12 isolates of an unknown pathotype from the Kyoto Prefecture, as well as 12 isolates of known pathotypes, including three single-spore lines. From the 12 isolates from Kyoto Prefecture, 11 were classified into either pathotype 2 (three isolates) or 4 (eight isolates). We designed 23 SSR markers based on the P. brassicae genome, of which 11 markers from intergenic regions showed polymorphisms in the 24 isolates. Many haploid isolates belonging to pathotypes 2 and 4 were monomorphic, and typical alleles were detected in some isolates not belonging to pathotype 4. Two bands were detected for eight SSR loci in five isolates, indicating that different genotypes were mixed in these isolates. We constructed a phylogram based on the 11 polymorphic SSRs. Pathotypes 2 and 4 formed a cluster, from which pathotypes 3 and 1 were successively placed. These results strongly suggest a close genetic relationship between isolates in pathotypes 2 and 4, consistent with our finding that isolates in these two pathotypes were found at one collection site. In combination with pathotype classification and other marker systems, the SSR markers can be used for more detailed analyses to improve the control of clubroot disease.     

Associated bacterial diversity of insecticide-susceptible and -resistant brown planthopper, <Emphasis Type="Italic">Nilaparvata lugens</Emphasis> (Homoptera: Delphacidae) analyzed by culture-dependent and -independent methods

Nilaparvata lugens (Stål) is one of the major pests of rice throughout tropical and temperate Asia. Indiscriminate use of insecticides for suppressing N. lugens has resulted in the development of resistance to multiple insecticide classes, causing frequent control failures in the field. Analysis of gut bacterial diversity within an insect host is the initial step towards understanding the ecological roles of the symbionts. Present study aimed to survey the bacterial diversity associated with laboratory-reared (insecticide-susceptible) and field-collected (insecticide-resistant) populations of N. lugens by culture-dependent and PCR-Denaturing Gradient Gel Electrophoresis (DGGE) methods. Seventeen bacterial isolates were obtained by the culture-dependent method. Molecular characterization using the 16S rRNA gene and phylogenetic analysis revealed that the isolates belonged to Firmicutes and Proteobacteria. Taxonomic assignment placed these isolates into seven families representing 10 genera. Enterobacteriaceae was the most dominant family with its occurrence in four out of the five populations studied. The DGGE profiles indicated a low complex gut bacteria associated with N. lugens with limited number of bands. The Shannon-Wiener index ranged from 0.898 in insecticide-susceptible population to 0.946–1.035 in resistant populations. Sequencing and phylogenetic analysis revealed that the DGGE bands belonged to Firmicutes, Proteobacteria and Bacteriodetes. Results of this study illustrated that gut bacterial community associated with N. lugens is dominated by Proteobacteria and Firmicutes. Present findings could provide the basis for future work on the possible role of the bacterial symbionts in insecticide resistance and to formulate potential resistance management strategies.     

The East-Mediterranean cereal leaf miner <Emphasis Type="Italic">Syringopais temperatella</Emphasis> Lederer and its larval parasitoids in Israel

Leaves of durum wheat infested with mines of the cereal leaf miner Syringopais temperatella Lederer (Lepidoptera: Scythridae) were collected from fields in Israel in spring 2016 and 2017. The parasitoids and moths reared from the leaf mines in the lab were identified and counted according to sex. The sex ratios of S. temperatella were 1:0.73 (♀ :♂) in 2016 and 1:0.41 in 2017. A cross-correlation analysis revealed that males appeared three days ahead of females (p<0.05). The following parasitoids were reared: Eulophidae (Cirrospilus vittatus Walker, Diglyphus chabrias (Walker), D. isaea (Walker), D. sensilis Yefremova, D. pusztensis (Erd?s & Novicky), Necremnus tidius (Walker), Neochrysocharis formosus (Westwood), Pnigalio gyamiensis Myartseva & Kurashev, P. pectinicornis Linnaeus), Pteromalidae (Norbanus sp.) and Braconidae (Habrobracon stabilis (Wesmail), Apanteles sp.); and Icheumonidae (Campoplex sp.). In all cases, the parasitoids emerged substantially prior to the mass appearance of the moths. The hatching dynamics of ecto- and endoparasitoids were also asynchronous. Most species of ectoparasitoids hatched three days ahead of the endoparasitoids (p<0.05). Syringopais temperatella recorded on clover revealed a similar dynamics of male appearance ahead of female, with a of female-to-male ratio of 1:0.25, and fewer parasitoid species.     

<Emphasis Type="Italic">RB</Emphasis> and <Emphasis Type="Italic">Ph</Emphasis> resistance genes in potato and tomato minimize risk for oospore production in the presence of mating pairs of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Composed mostly of fungivorous species, the genus Aphelenchoides also comprises 14 plant-parasitic species. The most common and devastating, A. besseyi, A. fragariae, A. ritzemabosi and A. subtenuis have been reported on more than 900 plant species. The combination of low inter-specific and high intra-specific morphological variability makes morphology-based identification extremely difficult within this genus, and has led to molecular tools being employed to ensure accurate diagnoses. rDNA markers are widely used for the identification of nematodes while the Cytochrome Oxidase I gene (COI) remains relatively unexplored despite its role as the standard barcode for almost all animal groups. To explore its suitability as a diagnostic tool, we studied a fragment of the mtCOI region of the four main plant-parasitic Aphelenchoides within a phylogenetic framework. We generated 69 mtCOI and 123 rDNA sequences of diverse Aphelenchoides taxa; 67 belong to the main plant-parasitic species including the first mtCOI sequence of A. fragariae and the first mtCOI and 28S sequences of A. subtenuis. mtCOI had a similar success rate for PCR amplification. Phylogenetic trees based on the three studied markers are largely in agreement with one another, validating their use for Aphelenchoides diagnosis; additionally, we were able to locate several misidentified sequences of plant-parasitic Aphelenchoides in existing databases. The concatenated analysis from the three markers resulted in a more robust insight into the phylogeny and evolution of Aphelenchoides, revealing that plant-parasitism has evolved independently at least three times within this genus, presumably from fungal-feeding ancestors.     

Histological observation of cucumber infected with <Emphasis Type="Italic">Corynespora cassiicola</Emphasis>

Erwinia amylovora is the causative agent of fire blight, which is a destructive bacterial disease of rosaceous plants. In Hungary Erwinia amylovora (Burrill) Winslow et al. was first detected in 1996. Since the appearance of fire blight, E. amylovora samples have been collected from different host plats from various geographic locations. A motif of eight nucleotides (ATTACAGA) is repeated 3–15 times in the PstI fragment of the pEa29 plasmid in Erwinia amylovora strains, and represents a valuable tool for strain classification. The number of short-sequence DNA repeats in plasmid pEa29 of 30 Hungarian isolates were determined by PCR assays and they ranged from five to ten. The SSR test is suitable for distinguishing the individual strains between the E. amylovora isolates. The examined isolates showed high pathogenicity on immature pear fruits. Several biochemical techniques, such as miniaturized API 20E, were applied on the samples. Differences were also revealed in microbiological assays like levan formation and colony morphology on semi-selective media. Examining the Hungarian Erwinia amylovora population by molecular analysis we can draw the conclusion that the population consists of different strains, which shows great diversity. E. amylovora is a widespread pathogen in Hungary, which is supported by the 30 strains isolated from various host plants from many parts of the country. The phenotypic diversity-evaluation of the E. amylovora strains showed, that they differ metabolically, like other plant pathogenic bacteria as reported by several authors. This is the first report on the diversity of E. amylovora strains isolated from Hungary.     

Molecular characterization of <Emphasis Type="Italic">Ditylenchus dipsaci</Emphasis> on Onion in Turkey

Ditylenchus dipsaci is a species complex including diploid and polyploid individuals. The onion race of D. dipsaci is a sensu stricto group and has a wide range of host spectrum. Identification of the D. dipsaci onion race is difficult using morphological and morphometrical methods. Species specific primers are mostly used in molecular approaches for identification of D. dipsaci populations. Fifty one morphologically selected Ditylenchus spp. populations from onion production areas in Turkey were subjected to molecular identification using four D. dipsaci species specific primer sets (PF1-PR1, PF2-PR2, DdpS1-rDNA2, DitNF1- rDNA2, H05-H06) targeting 5.8S and 18S rDNA, ITS1 and flanking ITS regions. Thirty nine percent of the nematode samples were positive with four primers tested, while four of the nematode samples gave specific bands with H05-H06 primers. Ditylenchus dipsaci sensu stricto was identified with specific primer sets in Adana, Hatay, Tekirdag, Bursa, Aksaray, Karaman, Eskisehir and Ankara provinces in Mediterranean, Trace, Aegean and Central Regions in Turkey.     

Outcome of sexual reproduction in the <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population in Estonian potato fields

In this study, the Estonian population of Phytophthora infestans was characterized with mating type, sensitivity to metalaxyl, virulence on 11 potato R-gene differentials and 12 SSR markers to show the outcome of potential sexual reproduction in the population. During the three years 2010–2012, 141 P. infestans isolates, collected from 23 potato fields, showed quite a high and stable frequency of the A2 mating type, 48% of the total population. In 87% of all sampled potato fields, both mating types were recorded, suggesting continuous sexual reproduction of P. infestans and possible oospore production. Metalaxyl-sensitive isolates prevailed in all three years (68 out of 99 isolates). Amongst the 95 isolates tested, 51 virulence races were found. The race structure was diverse, and most pathotypes were unique, appearing only once; the two most common pathotypes, 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11, comprised 35% of the population. The P. infestans population was genetically highly diverse and most of the multilocus genotypes (MLGs) appeared only once. Furthermore, all of the MLGs appeared in only one of the three sampling years. Our results confirm that the high diversity in the Estonian P. infestans population is most likely the result of frequent sexual reproduction, which benefits the survival, adaptability and diversity of the pathogen in the climate of North-Eastern Europe.     

Morphological and molecular characterization of <Emphasis Type="Italic">Diaporthe (</Emphasis>anamorph <Emphasis Type="Italic">Phomopsis)</Emphasis> complex and pathogenicity of <Emphasis Type="Italic">Diaporthe aspalathi</Emphasis> isolates causing stem canker in soybean

The complex of Diaporthe (asexual morph) species occurring on soybean constitutes an important pathogenic group associated with diseases such as pod and stem blight, seed decay and stem canker. Stem canker, caused by Diaporthe aspalathi, has been reported as the most aggressive form of canker and its occurrence has limited soybean crop productivity in the southern United States. The main form of pathogen control is the use of stem canker resistant soybean varieties. In this study, strains of Diaporthe and Phomopsis were isolated from stem and seeds of soybean in different locations in South America during the years 1989–2014. Genomic DNA from 26 isolates were analyzed by PCR-restriction fragment length polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP) techniques, and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Phomopsis longicolla, D. phaseolorum var. sojae, D. caulivora and D. aspalathi. An analysis of the pathogenicity of 13 isolates of D. aspalathi inoculated in soybean genotypes carrying different resistance genes to stem canker (Rdm1, Rdm2, Rdm3, Rdm4, Rdm5 and Rdm?) enabled us to identify the occurrence of at least three races of D. aspalathi occurring in Brazil. Among the isolates identified as D. aspalathi, both molecular and phenotypic analyses showed clustering depending on the date of collection and pathogenicity, which revealed the existence of variability of the pathogen.     

Efficacy of a formulated product containing <Emphasis Type="Italic">Quillaja saponaria</Emphasis> plant extracts for the control of root-knot nematodes

The nematicidal effect of a formulated product containing extract from Quillaja saponaria was evaluated against the root-knot nematodes. The product QL Agri® 35 (QL) was tested to record the effect on second stage juveniles motility, egg hatch and also against field populations in greenhouse experiments contacted in three different locations of Greece. Convulsive movement of second stage juveniles of Meloidogyne incognita was recorded after exposure for 8 days at a series of doses, while the most paralyzed juveniles were counted at the dose of 8 mg l^?1. There was also a gradual decrease in the number of juveniles emerging from egg masses of the same nematode species when the dose of Q. saponaria was increased from 0 to 8 mg l^?1. In greenhouse experiments, the use of Q. saponaria could control root-knot nematodes and prevent nematodes increase in soil. The present study demonstrates that the use of Q. saponaria extract has the ability to control root-knot nematodes. Control given by Q. saponaria in field populations infecting cucumber was similar to that of cadusafos (Rugby®) and oxamyl (Vydate®) under the tested dosages and the specific conditions of the experiments.