Pathogenicity of Pythium species to maize

Pythium isolates from diseased and dead bait plants of maize and cress grown in compost or various soils (maize fields, parkland under deciduous trees, grassland) were characterised and tested for pathogenicity to maize (Zea mays L.). In pot tests performed under controlled conditions, pathogenicity of the isolates to maize was apparent by reduction of root and shoot growth, whereas damping-off of maize seedlings was less frequent. Contrarily, pea seedlings were killed by pathogenic Pythium isolates. Pythium isolates from diseased maize seedlings and pathogenic strains from other gramineous plants (P. phragmitis, P. aff.phragmitis, P. catenulatum) were not necessarily more virulent to maize compared to isolates originating from dicotyledonous plants (cress). The most virulent isolates originated from compost and caused a reduction of maize shoot growth of up to 60%. Phylogenetic analysis revealed that they were very closely related to P. ultimum var. ultimum and P. arrhenomanes, respectively. Isolates originating from maize fields, grassland and parkland under deciduous trees, a reference culture of P. arrhenomanes and strains of P. phragmitis, P. aff. phragmitis and P. catenulatum with known pathogenicity on reed were non-pathogenic on maize. Isolates from compost, and from maize fields generally had a higher temperature optimum for mycelial growth (30 °C) and a faster growth rate (1.5–2.0 mm h−1) compared to the isolates from parkland under deciduous trees and grassland soil (20–25 °C, ~1.0 mm h−1), respectively. This study indicates a potential impact of pathogenic Pythium on maize plants even in the absence of visible symptoms.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

Methods to Study the PVY Population in the Potato

The PVY population in the potato has been studied continuously using tobacco bait plants in potato fields at Młochów since 1980 at two-year intervals and in potato tuber samples collected from different regions of Poland since 2001 yearly. The paper presents the combined biological, serological and molecular assays for PVY identification and strain classification. Biologically, PVY strains are defined with respect to their ability to elicit hypersensitive resistance (HR) mediated by N genes in differential potato cultivars (King Edward, Desiree and Pentland Ivory) and to symptoms in the tobacco (cultivar Samsun). Serologically, an ELISA assay based on polyclonal or monoclonal cocktail antibodies recognizes all PVY strain types, while the specific monoclonal antibodies help to recognize PVYN or PVYO/PVYCstrains. Multiplex RT-PCR, Real-time RTqPCR and sequencing-based assays are used to define the PVY genome structure. In the Polish population of PVY, the strains PVYO, PVYNTN, PVYN-Wi, PVYZ-NTN and PVYEwere identified, while the PVYCstrain was not detected.

Polymyxa graminis Isolates from Australia: Identification in Wheat Roots and Soil, Molecular Characterization, and Wide Genetic Diversity

Polymyxa graminis is an obligate parasite of roots and an important vector of viruses that damage cereal crops in different parts of the world. In 2011 and 2012, P. graminis was identified infecting 11 wheat root samples from three widely dispersed locations in southwest Australia. Its presence was detected by polymerase chain reaction (PCR) and confirmed by DNA sequencing of the transcribed regions of its ribosomal RNA genes (rDNA) and observing sporosori of characteristic morphology and size in stained wheat roots. Also, when soil samples were collected from two locations where P. graminis was found and wheat bait plants grown in them, P. graminis was detected in their roots by PCR. Ribosomal DNA sequences of six southwest Australian isolates were obtained from wheat roots, and one northeast Australian isolate from barley roots. When these seven P. graminis sequences were compared with others from GenBank by phylogenetic analysis, three southwest Australian isolates were classified as P. graminis f. sp. temperata (ribotypes Ia and Ib), and three as f. sp. tepida (ribotypes IIa and IIb). P. graminis f. sp. temperata and tepida both occur in temperate growing regions of other continents and are associated with transmission of soil-borne viruses to cereal crops. The P. graminis isolate from northeast Australia was sufficiently distinct from the five existing sequence groups for it to be placed into a newly proposed grouping, ribotype VI, which also included an isolate from tropical West Africa. However, when randomly collected wheat leaf samples from 39 field crops from 27 widely dispersed locations, 21 individual wheat plant samples collected from low lying areas within 21 fields at 11 locations, and wheat bait plants growing in five soil samples from two locations were tested by reverse transcription (RT)-PCR for the presence of Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic virus, and furoviruses in general, no virus infection was detected. These findings suggest at least three P. graminis introductions into Australia, and the occurrence of f. sp. temperata (ribotype I) and f. sp. tepida (ribotype II) suggests that, if not already present, soil-borne cereal viruses are likely to become established should they become introduced to the continent in the future.

First Report of Olpidium bornovanus and O. virulentus on Melon in Italy

A survey for the presence of Olpidium spp. on melon (Cucumis melo L.) was conducted during the beginning of 2013 in central Italy in an unheated greenhouse, located in the melon-producing coastal area of north Latium (central Italy, Viterbo Province) (42°23′09.31″N, 11°30′46.10″E) with a history of monosporascus root rot and vine decline (MRRVD). For this aim, 10 soil samples were collected adjacent to the roots of plants symptomatic of MRRVD, represented by root lesions and rots and loss of smaller feeder roots. Olpidium was baited from collected infested soil by growing melon (cv. Dinero) plants for 45 days. Bait plants grown in sterilized soil were used as negative controls. All the baited melon roots were analyzed by morphological and molecular methods. For the morphological analysis, feeder roots were clarified in a 1.5% KOH solution for 24 h (2) and observed under a light microscope to record the presence or absence of sporangia and resting spores of Olpidium spp., which were observed in baited melon plants grown in infested soil and not in control roots. In particular, stellate resting spores were referred to as O. virulentus because this species cannot be distinguished from O. brassicae, which does not colonize melon. O. bornovanus had smooth-walled resting spores with a honeycomb-like pattern (2). For molecular analysis, DNA was extracted from 21 melon roots and tested by multiplex PCR to confirm Olpidium spp. identification (2). Based on molecular identification, O. virulentus was identified in 40% of samples, and O. bornovanus was identified in 10%. There were no mixed infections in the same sample. Two amplified PCR products, corresponding to O. bornovanus and O. virulentus expected fragment sizes of 977 and 579 bp respectively, were sequenced (GenBank Accession Nos. KF661295 and KF661296). BLAST analysis of the sequences showed 99% nucleotide identity with O. bornovanus isolate CH from Japan collected in melon roots (AB205215) and O. virulentus isolate HY-1 from Japan collected in lettuce roots as reported by Sasaya and Koganezawa (3) (AB205204, formerly O. brassicae). At the end of the experiment, the root systems of all inoculated plants appeared brown, whereas neither symptoms nor sporangia and resting spores were observed in roots of control plants. Olpidium spp. are root-infecting plant pathogens of melon (4), acting as vectors of Melon necrotic spot virus (MNSV) and other destructive plant viruses (1). Moreover, they are directly involved in the induction of germination of ascospores of Monosporascus cannonballus, the causal agent of MRRVD of cucurbits (4). To our knowledge, this is the first report of O. virulentus and O. bornovanus on melon in Italy. References: (1) A. Alfaro-Fernández et al. J. Phytopathol. 91:1250, 2009. (2) J. A. Herrera-Vásquez et al. Mycol. Res. 113:602, 2009. (3) T. Sasaya and H. Koganezawa. J. Gen. Plant Pathol. 72:20, 2006. (4) M. E. Stanghellini and I. J. Misaghi. Phytopathology 101:794, 2011.

Infestation of Polish Agricultural Soils by Plasmodiophora Brassicae Along The Polish-Ukrainian Border

There has been a rapid, worldwide increase in oilseed rape production that has resulted in enormous intensification of oilseed rape cultivation, leading to tight rotations. This in turn, has caused an accumulation of pests as well as foliar and soil-borne diseases. Recently, clubroot has become one of the biggest concerns of oilseed rape growers. Clubroot is caused by the soil-borne protist Plasmodiophora brassicae Woronin. The pathogen may be present in groundwater, lakes, and irrigation water used in sprinkling systems. It can be easily transmitted from one field to another not only by water, but also by soil particles and dust transmitted by wind and on machinery. The aim of our overall study was to check for P. brassicae infestation of Polish agricultural soils. This paper presents the 2012 results of a study performed along the Polish-Ukrainian border in two provinces: Lublin (Lubelskie Voivodeship) and the Carpathian Foothills (Podkarpackie Voivodeship), in south-east Poland. Monitoring was done in 11 counties, including nine rural and two municipal ones. In total, 40 samples were collected, out of which 36 were collected from fields located in rural areas and four from municipal areas, with two per municipal region. Each sample was collected at 8-10 sites per field, using a soil auger. The biotest to detect the presence of P. brassicae was done under greenhouse conditions using seedlings of the susceptible Brassicas: B. rapa ssp. pekinensis and the Polish variety of oilseed rape B. napus cv. Monolit. Susceptible plants grown in heavily infested soils produced galls on their roots. A county was regarded as free from the pathogen, if none of the bait plants became infected. The pathogen was found in three out of 40 fields monitored (7.5%) in the Carpathian Foothill region. The fields were located in two rural counties. The pathogen was not found in Lublin province, and was also not detected in any of the municipal counties. The detection with a biotest was fully confirmed by PCR-based molecular detection of P. brassicae DNA in soil samples.

Longidoridae and nepoviruses in Bulgaria and Slovenia

Data on the distribution of Longidoridae and nepoviruses in Bulgaria and Slovenia are summarized. Six species of Longidorus (L. apulus, L. attenuatus, L. arthensis, L. fasciatus, L. elongatus, L. macrosoma), one Paralongidorus species (P. maximus) and three Xiphinema species (X. diversicaudatum, X. index, X. rivesi) are known as natural vectors of nine nepoviruses in Europe. Currently, 10 and 13 species of Xiphinema; 6 and 15 of Longidorus are reported to occur in Slovenia and Bulgaria, respectively. Paralongidorus maximus has been reported only in Bulgaria. Among the virus vector species X. index, X. diversicaudatum and L. elongatus occur in both countries, X. rivesi only in Slovenia and L. attenuatus, L. macrosoma, X. italiae and P. maximus only in Bulgaria. A report of X. index and Grapevine fanleaf virus (GFLV) in Bulgaria was related to transgenic grape tolerance to the same virus. Nepoviruses have been reported from Slovenia, but despite an evident relationship in the occurrence of GFLV and X. index in several vineyards the only laboratory proven transmission is that of TRSV and ToRSV to bait plants by a Slovenian population of X. rivesi.