Population Structure and Management of Podosphaera pannosa Associated with Peach Powdery Mildew in Oman

In 2004, severe powdery mildew infection on peach occurred in Al‐Jabal Al‐Akdhar, Oman, and resulted in substantial yield losses to growers. This study was conducted to investigate occurrence, causal agents, genetic diversity and efficacy of azoxystrobin in management of this disease. Powdery mildew was observed on all farms and peach trees in Al‐Jabal Al‐Akdhar. Disease symptoms were first observed on shoots in April, followed by appearance on fruits. Disease severity reached its peak between May and June. Morphological and molecular identification of 22 powdery mildew isolates indicated that all belong to Podosphaera pannosa. Podosphaera pannosa reproduced the same symptoms upon inoculation on peach leaves. Amplified fragment length polymorphisms analysis of 35 isolates of P. pannosa from five different villages using four primer pair combinations produced 688 polymorphic loci and 35 different genotypes. Populations of P. pannosa were found to have low levels of gene diversity (H = 0.1858), which suggests that P. pannosa has been recently introduced into Al‐Jabal Al‐Akdhar. Analysis of molecular variance showed low levels of genetic differentiation among populations from the different villages, implying the introduction of P. pannosa into the different villages via common sources as well as frequent movement of pathogen inoculum among the different villages. Evaluating the efficacy of azoxystrobin showed that azoxystrobin is as efficacious as thiophanate‐methyl in managing the disease, with sulphur being the least efficacious. The study is the first to report the presence of P. pannosa in Oman. Also reported are its genetic diversity and its management under commercial conditions.     

Sensitivity of Zymoseptoria tritici Isolates from Tunisia to Pyraclostrobin,Fluxapyroxad, Epoxiconazole,Metconazole, Prochloraz and Tebuconazole

Sensitivity of 159 isolates of Zymoseptoria tritici collected from durum wheat fields in Tunisia in 2012 was analysed towards pyraclostrobin, fluxapyroxad, epoxiconazole, metconazole, prochloraz and tebuconazole using microtiter tests. All isolates were found to be highly sensitive to pyraclostrobin with EC₅₀ <0.01 mg/l with the exception of three isolates from the same field with higher EC₅₀ values (>0.5 mg/l). These three isolates carried a mutation in the cytochrome b gene encoding the G143A substitution. This is the first report of quinone outside inhibitors (QoI) resistance in Z. tritici in Tunisia. Sensitivity towards r fluxapyroxad was in a narrow range with EC₅₀ values ranging between 0.013 and 0.125 mg/l, which can serve as baseline sensitivity data for the future. Demethylation inhibitors sensitivity varied across a broad range with the data indicating a slight shift in sensitivity when compared to a previous study on the 2010 population. No highly sensitive strains were isolated from samples from fields, which had received three or four DMI applications.     

Occurrence of fenhexamid resistance in Botrytis cinerea from greenhouse strawberries in China

This study assessed the fenhexamid sensitivity of 143 Botrytis cinerea isolates collected from greenhouse strawberries in five regions of China between 2012 and 2013, and identified four isolates with moderate levels of resistance: two from the Xinjiang Uygur Autonomous Region and two from Hebei Province. The baseline fenhexamid sensitivity of B. cinerea exhibited a unimodal distribution with a mean EC₅₀ value of 0.20 ± 0.10 μg/ml (SD). The EC₅₀ values of the fenhexamid‐resistant isolates ranged from 0.05 to 0.40 μg/ml. Molecular analysis of the fenhexamid target gene erg27 revealed that the resistant isolates collected from Xinjiang (163‐6 and 163‐22) contained three mutations that led to amino acid changes (V365A, E368D and A378T) known to be associated with fenhexamid resistance, but that the isolates from Hebei lacked any mutations, indicating that an alternative mechanism could be responsible for their resistance. Most of the biological characteristics of the fenhexamid‐resistant isolates, such as mycelial growth, sclerotia production and pathogenicity, did not significantly differ from those of the sensitive ones (p ≤ .05), but it was noted that some of the resistant isolates exhibited reduced rates of sporulation and spore germination. In addition, the resistant isolates exhibited lower osmotic sensitivity than the sensitive ones. The study found no evidence of cross‐resistance with other fungicides, but that there was negative cross‐resistance with procymidone, iprodione, carbendazim and pyraclostrobin, which indicates that the inclusion of these fungicides within an integrated pest management (IPM) programme could help to minimize the risk of fenhexamid resistance developing in B. cinerea.     

Long range control circuits within mitochondria and between nucleus and mitochondria

Summary In the preceding paper of this series (Dujardin et al. 1980a) we described general methods of selecting and genetically characterizing suppressor mutations that restore the respiratory capacity of mit ^- mitochondrial mutations. Two dominant nuclear (NAM1-1 and NAM2-1) and one mitochondrial (mim2-1) suppressors are more extensively studied in this paper. We have analysed the action spectrum of these suppressors on 433 mit ^- mutations located in various mitochondrial genes and found that they preferentially alleviate the effects of mutations located within intron open reading frames of the cob-box gene. We conclude that these suppressors permit the maturation of cytochrome b mRNA by restoring the synthesis of intron encoded protein(s) catalytically involved in splicing i.e. mRNA-maturase(s) (cf. Lazowska et al. 1980). NAM1-1 is allele specific and gene non-specific: it suppresses mutations located within different introns. NAM2-1 and mim2-1 are intron-specific: they suppress mutations all located in the same (box7) intron of the cobbox gene. Analyses of cytochrome absorption spectra and mitochondrial translation products of cells in which the suppressors are associated with various other mit ^- mutations show that the suppressors restore cytochrome b and/or cytochrome oxidase (cox 1) synthesis, as expected from their growth phenotype. This suppression is, however, only partial: some new polypeptides characteristic of the mit ^- mutations can be still detected in the presence of suppressor. Interestingly enough when box7 specific suppressors NAM2-1 and mim2-1 are associated with a complete cob-box deletion (leading to a total deficiency of cytochrome b and oxidase) partial restoration of cox I synthesis is observed while cytochrome b is still totally absent. These results show that in strains carrying NAM2-1 or mim2-1 the presence of cytochrome b gene is no longer required for the expression of the oxi3 gene pointing out to the possibility of a mutational switch-on of silent genes, whether mitochondrial, mim2-1, or nuclear, NAM2-1. This switch-on would permit the synthesis of an active maturase acting as a substitute for the box7 maturase in order to splice the cytochrome b and oxidase mRNAs.     

Restriction to large‐scale gene flow vs. regional panmixia among cold seep Escarpia spp. (Polychaeta,Siboglinidae)

The history of colonization and dispersal in fauna distributed among deep‐sea chemosynthetic ecosystems remains enigmatic and poorly understood because of an inability to mark and track individuals. A combination of molecular, morphological and environmental data improves understanding of spatial and temporal scales at which panmixia, disruption of gene flow or even speciation may occur. Vestimentiferan tubeworms of the genus Escarpia are important components of deep ‐sea cold seep ecosystems, as they provide long‐term habitat for many other taxa. Three species of Escarpia, Escarpia spicata [Gulf of California (GoC)], Escarpia laminata [Gulf of Mexico (GoM)] and Escarpia southwardae (West African Cold Seeps), have been described based on morphology, but are not discriminated through the use of mitochondrial markers (cytochrome oxidase subunit 1; large ribosomal subunit rDNA, 16S; cytochrome b). Here, we also sequenced the exon‐primed intron‐crossing Haemoglobin subunit B2 intron and genotyped 28 microsatellites to (i) determine the level of genetic differentiation, if any, among the three geographically separated entities and (ii) identify possible population structure at the regional scale within the GoM and West Africa. Results at the global scale support the occurrence of three genetically distinct groups. At the regional scale among eight sampling sites of E. laminata (n = 129) and among three sampling sites of E. southwardae (n = 80), no population structure was detected. These findings suggest that despite the patchiness and isolation of seep habitats, connectivity is high on regional scales.     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Isolation and characterization of RDX-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> species from a contaminated aquifer

Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel’s coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 μM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l⁻¹) while the T9N isolate’s activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.     

Taxonomic composition and ploidy level among European water frogs (Anura: Ranidae: Pelophylax) in eastern Hungary

The western Palaearctic water frogs in the genus Pelophylax comprise several distinct species and three hybridogenetic hybrid forms. In this study, we focus on the Pelophylax esculentus complex, which consists of two sexual species, Pelophylax ridibundus and Pelophylax lessonae, and their hybridogenetic hybrid, Pelophylax esculentus. Specifically, we investigated taxonomic composition and ploidy level of water frogs sampled in three different types of wetland habitats in the Hortobágy National Park (HNP), eastern Hungary. Using variation in serum albumin intron 1 (SAI‐1) and 15 microsatellite loci, we detected the presence of all members of the P. esculentus complex in the studied localities. In one locality, all three taxa occurred syntopically, while in others water frog populations consisted of P. ridibundus and P. esculentus exclusively. The genomic composition of the 63 examined hybrid specimens analysed with microsatellites showed the occurrence of diploid genotypes only. We used a population genetic approach (allelic richness, gene diversity, multilocus genotypes and multilocus disequilibrium) to infer the breeding system of water frogs at HNP. Our data indicate that at least in two populations, hybrids form gametes with clonally transmitted P. ridibundus genome and produce a new hybrid generation by mating with P. lessonae.     

KIT and melanoma predisposition in pigs: sequence variants and association analysis

KIT mutations have been detected in different cancer subtypes, including melanoma. The gene also has been extensively studied in farm animals for its prominent role in coat color. The present work aimed at detecting KIT variants in a porcine model of cutaneous melanoma, the melanoblastoma‐bearing Libechov Minipig (MeLiM). By sequencing exons and intron borders, 36 SNPs and one indel were identified. Of 10 coding SNPs, three were non‐synonymous mutations, likely to affect the protein conformation. A promising variant, located in exon 19 (p.Val870Ala), was genotyped in a MeLiM × Duroc cross, and an association analysis was conducted on several melanoma‐related traits. This variant showed a significant association with melanoma development, tumor ulceration and cutaneous invasion. In conclusion, although the KIT gene would not be a major causal gene for melanoma development in pig, its genetic variation could be influencing this trait.     

Invasion promotes invasion: Facilitation of C3 perennial grass dominance in mixed C3/C4 grassland by an invasive C3 woody sprouter (Prosopis glandulosa)

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Novel variant Pro143Ala in <Emphasis Type="Italic">HTRA2</Emphasis> contributes to Parkinson’s disease by inducing hyperphosphorylation of HTRA2 protein in mitochondria

Mutations in the gene encoding the mitochondrial protein high temperature requirement A2 (HTRA2) are inconsistently associated with a risk of Parkinson’s disease (PD). We assessed the presence of HTRA2 mutations among patients with PD and performed functional assay of identified mutations or variants. Among the total 1,373 subjects, the entire HTRA2 coding region was sequenced in 113 early-onset PD (EOPD), 20 familial PD patients and 150 control subjects. An additional 390 sporadic late-onset PD patients and 700 controls were subsequently screened to validate possible mutations found in the first set. We identified two novel heterozygous variants, c.427C > G (Pro143Ala) and c.906 +3 G > A, in 2 (1.5%) EOPD patients. The missense variant, Pro143Ala, was also observed in one late-onset PD patient but was absent in total 850 control subjects (relative risk 2.3, 95% CI 1.5–2.8, P = 0.04). Expressing Pro143Ala variant of HTRA2 in primary dopaminergic neurons causes neurite degeneration. Following exposure to rotenone, the ultra-structural mitochondrial abnormality, the percentage of mitochondrial dysfunction and apoptosis in cells carrying the HTRA2 Pro143Ala variant was significantly higher than wild-type cells. Mechanistically, protein level of phosphorylated HTRA2 was increased in cells carrying the Pro143Ala variant, suggesting Pro143Ala variant promotes HTRA2 phosphorylation with resultant mitochondrial dysfunction. Our results support a biologically relevant role of HTRA2 in PD susceptibility in Taiwanese. Further large-scale association studies are warranted to confirm the role of HTRA2 Pro143Ala variant in the risk of PD.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Phytophthora Species Infecting Hardy Ornamentals in Nurseries and the Managed Environment in Scotland

Between 2002 and the end of 2009, more than 4000 samples from hardy ornamental plants, collected in surveys for Phytophthora ramorum, were examined to establish the occurrence and diversity of Phytophthora species in Scotland. The samples were gathered from more than 77 plant genera in nurseries, gardens and amenity landscapes. Fifteen different Phytophthora spp. were isolated and identified either by polymerase chain reaction (PCR) or by sequencing of the ITS1, 5.8S subunit and ITS2 region of the ribosomal RNA gene. The most widespread Phytophthora spp. were P. ramorum and P. syringae, followed by P. cactorum, P. kernoviae, P. plurivora, P. cambivora, P. citrophthora, P. taxon ‘Pgchlamydo’, P. pseudosyringae and some single isolates of P. cinnamomi, P. cryptogea, P. gonapodyides, P. nicotianae and P. hibernalis. One isolate did not match any known species. In relation to the number of samples, Phytophthora was found more frequently in trade premises than in gardens or amenity landscapes and the species diversity was higher, highlighting the risks involved in plant trade.     

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole‐genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.