Guillermo A. Galván

Molecular diversity of arbuscular mycorrhizal fungi in onion roots from organic and conventional farming systems in the Netherlands

Diversity and colonization levels of naturally occurring arbuscular mycorrhizal fungi (AMF) in onion roots were studied to compare organic and conventional farming systems in the Netherlands. In 2004, 20 onion fields were sampled in a balanced survey between farming systems and between two regions, namely, Zeeland and Flevoland. In 2005, nine conventional and ten organic fields were additionally surveyed in Flevoland. AMF phylotypes were identified by rDNA sequencing. All plants were colonized, with 60% for arbuscular colonization and 84% for hyphal colonization as grand means. In Zeeland, onion roots from organic fields had higher fractional colonization levels than those from conventional fields. Onion yields in conventional farming were positively correlated with colonization level. Overall, 14 AMF phylotypes were identified. The number of phylotypes per field ranged from one to six. Two phylotypes associated with the Glomus mosseae–coronatum and the G. caledonium–geosporum species complexes were the most abundant, whereas other phylotypes were infrequently found. Organic and conventional farming systems had similar number of phylotypes per field and Shannon diversity indices. A few organic and conventional fields had larger number of phylotypes, including phylotypes associated with the genera Glomus-B, Archaeospora, and Paraglomus. This suggests that farming systems as such did not influence AMF diversity, but rather specific environmental conditions or agricultural practices.

Genetic variation among Fusarium isolates from onion, and resistance to Fusarium basal rot in related Allium species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.

In vitro antimicrobial activity of different accessions ofSolanum commersonii Dun. from Uruguay

SummaryThe in vitro antimicrobial activity of extracts from accessions ofSolanum commersonii Dun. collected in the south of Uruguay was investigated against five microorganisms including the pathogenRalstonia solanacearum. A total of 30 extracts corresponding to organic and aqueous extracts were studied. Interestingly, most of the positive results for growth inhibition were againstR. solanacearum. The extracts were also analyzed for the presence of glycoalkaloids and lectins. Six of the ten aqueous extracts showed lectin presence and a wide variation in the type and amounts of glycoalkaloids, was found. Results indicate that there is no clear relationship between the antimicrobial activity against the five microorganisms screened and the presence or amounts of lectins and glycoalkaloids, traditionally regarded as possible antimicrobial metabolites in theSolanum genus, which indicates the presence of as yet unidentified antimicrobial compounds.

Quantitative studies on downy mildew (Peronospora destructor Berk. Casp.) affecting onion seed production in southern Uruguay

Onion downy mildew (Dm) symptoms and damage on seed production fields in southern Uruguay were quantified during two seasons as the progress of incidence, severity, and as the effect of the level of seed-stalks infections on seed yield and quality. In addition, the effects of two plantation dates and two plant densities on Dm were studied in a factorial experiment. Maximum incidence along the season ranged from 15 to 65% in four commercial fields in 2005 and two fields in 2006. Maximum severity ranged from 4.5 to 9.3% of leaf area affected in 2005, and 0.35 to 1.17% in 2006. Whereas Dm incidence varied among studied fields, disease progress as Dm severity varied mainly between studied years. Crop rotation, crop vigour and plant density were identified as major factors affecting Dm variation in the field. Intensive fungicide schedules did not control Dm when other conditions favoured the disease. A high level of Dm severity defined as large necklace spots on seed-stalks significantly reduced seed yield in comparison with healthy seed-stalks in 2005, as well as seed yield and germination in 2006. Weight of 1,000 seeds was not significantly reduced by Dm infections on seed-stalks. Six genera of fungi were recovered from seeds harvested on highly infected seed-stalks (Stemphylium, Fusarium, Alternaria, Penicillium, Aspergillus and Botrytis), but not Peronospora. Late planting date and low plant density had significantly lower Dm incidence and severity. This finding questioned the early planting dates and high densities previously recommended in order to achieve high yielding crops. The combination of several practices to reduce initial inoculum, susceptibility status of the host, and environmental conditions promoting the disease is discussed as forming a basis for effective disease control.

Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection

Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after R. solanacearum infection, including hyperplasic activity of conductor tissue, tylose production, callose and lignin deposition, and accumulation of reactive oxygen species. This study highlights the potential of the identified tolerant interspecific potato clones as valuable genetic resources for potato-breeding programs and leads to a better understanding of resistance against R. solanacearum in potato.

Age-related resistance to Fusarium oxysporum f. sp. cepae and associated enzymatic changes in seedlings of Allium cepa and A. fistulosum

This research analysed the response of onion (Allium cepa) and A. fistulosum against Fusarium oxysporum f. sp. cepae (Foc) isolates and the associated changes in peroxidase, β-1,3-glucanase and chitinase activities. The response of A. cepa and A. fistulosum at different stages of seedling development were also evaluated. Several seedling tests were performed, and disease symptoms were evaluated 12-14 days after inoculation. Allium fistulosum behaved as more resistant than A. cepa cultivars by exposition to the most aggressive Foc isolates at sowing date. Increased levels of peroxidase and glucanase activities were found in the A. cepa and A. fistulosum seedlings exposed to the pathogen, and were positively correlated with disease symptoms. For chitinase activity, this correlation was found only for A. cepa. Two peroxidase isoforms were found to be specific for A. fistulosum roots after inoculation and could be involved in resistance. The inoculation at 7, 14 and 42 days after sowing showed that both host species were resistant to Foc, proving that onion susceptibility decreased promptly after germination . However, an increase in peroxidase and glucanase activities in 7-and 14-day-old inoculated seedling was detected only for A. cepa, suggesting an earlier acquisition of resistance in A. fistulosum.

First Report of Iris yellow spot virus on Onion in Uruguay

From October to December 2005, onion (Allium cepa) plants in seed-production fields in south Uruguay (Canelones) had symptoms suggestive of those caused by Iris yellow spot virus (IYSV; genus Tospovirus, family Bunyaviridae). Symptoms included diamond-shaped lesions on seed stalks (scapes), each 1 to 5 cm long with a necrotic border, green center, and sometimes a second necrotic area in the center of the diamond (2,3). Necrotic lesions with more irregular shape were also associated with diseased plants. In 2006, scape samples with these symptoms were collected from four onion seed crops and assayed for IYSV using an IYSV-specific antiserum (Agdia Inc., Elkhart, IN) in a double-antibody sandwich-ELISA. IYSV was detected in all four onion seed crops monitored in 2006. IYSV incidence, expressed as the number of plants with symptoms, ranged from <1% (1 of 120 plants evaluated) to 20% (24 of 120 plants). Two fields were monitored in 2007, in which IYSV incidence increased from 2 and 3% in October to 7% (198 of 2,768 plants) and 40% (253 of 638 plants) in December, respectively. The highest incidence was observed in the same farm in 2006 and 2007. Scapes were sampled from the field with the highest incidence of symptoms in 2007 and tested for IYSV with IYSV-specific primers (3). Total RNA was extracted from 100 mg of symptomatic tissue, with green tissue adjacent to typical lesions, following a Trizol-based protocol (1). A reverse transcriptase-PCR assay with nucleocapsid gene-specific primers was used (3). A PCR product of approximately 26 bp was obtained, coincident with the expected length for IYSV. The PCR product was cloned and sequenced. The tospovirus N sequence of the isolate in Uruguay (Accession No. GU550518) had maximum identity (97%) with an Australian IYSV isolate (Accession No. AY345227), and >87% identity only with IYSV N protein sequences in GenBank. Because of the presence of IYSV in Brazil, Chile, and Peru, this first documentation, to our knowledge, of IYSV in onion crops in Uruguay suggests that the threat of IYSV to onion is increasing in South America. References: (1) P. Chomczynski and K. Mackey. Biotechniques 19:942, 1995. (2) D. H. Gent et al. Plant Dis. 90:1468, 2006. (3) H. R. Pappu et al. Plant Dis. 92:588, 2008.