Immunolocalization of <Emphasis Type="Italic">Pepper mild mottle virus</Emphasis> in developing seeds and seedlings of <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The location of Pepper mild mottle virus (PMMoV) within seeds as they developed on inoculated seedlings of pepper (Capsicum annuum) was followed over time by detecting the viral coat protein using immunofluorescence microscopy. Seedlings were inoculated with PMMoV when the flower buds on the first and second branching nodes were in bloom. Fluorescence indicating the presence of PMMoV was first observed around immature seeds and placentas in the ovaries on the fourth branching node at 20 days post-anthesis (20 DPA), which corresponded to 39 days post-inoculation (39 DPI). The area with fluorescence gradually expanded from the placenta into the integument and the parenchyma, and finally reached the tip of the immature seeds by 34 DPA (53 DPI). The embryo or endosperm beyond the endothelium never fluoresced during the experiment [i.e., ending at 81 DPA (102 DPI)]. For visualizing viral routes of invasion from seeds into new seedlings, PMMoV-infected C. annuum seeds that were heterozygous for the L ³ tobamovirus-resistance gene were sown in soil at 30°C. After ~2 weeks, the cotyledon developed virally induced necrosis. These findings shed light on the infection cycle of PMMoV through vertical transmission in C. annuum.     

Quantification of <Emphasis Type="Italic">Pyrenophora graminea</Emphasis> in barley seed using real-time PCR

A real-time PCR assay was designed to quantify seed-borne infection of Pyrenophora graminea in barley (Hordeum vulgare). Conventional tests such as the freezing blotter method cannot distinguish P. graminea from the closely related P. teres. The seed infection threshold for P. graminea is lower than the one for P. teres and is therefore applied for both species although P. graminea may be absent. This results in unnecessary rejections of seed lots. PCR primers and a TaqMan probe were designed to target a P. graminea-specific DNA sequence. The potential of the real-time PCR assay for quantifying seed-borne infection of P. graminea was investigated by examining seed lots harvested from P. graminea-infected fields. The major part (84%) of the variation in the amount of P. graminea DNA measured by real-time PCR could be attributed to variation between seed lots while only about 8% was due to variation within seed lots. DNA quantities of P. graminea were positively correlated with seed infection incidence detected by the freezing blotter method as well as with the infection incidence of plants examined in the greenhouse. Both correlations were highly significant (P < 0.001) but the DNA quantities accounted only for 59% (R ² = 0.59) and 56% (R ² = 0.56), respectively, of the variation in the results obtained by the two conventional methods. Seed lots of varieties resistant to P. graminea contained considerable amounts of P. graminea DNA but showed no or only few leaf symptoms in the greenhouse test suggesting that the recommended seed infection thresholds could be raised for resistant varieties.     

Cross-pathogenicity of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> between potato and sunflower

This study examined cross-pathogenicity of the soilborne pathogen Verticillium dahliae between potato and sunflower. Four week-old potato and sunflower seedlings were inoculated with ten isolates from each of the two host species. Potato cultivars (Kennebec, susceptible, and Ranger Russet, moderately resistant) and sunflower hybrids (IS8048, susceptible, and 6946, moderately resistant) were assessed for disease severity and percent infection at 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after inoculation (w.a.i), and for vascular discolouration at 6 w.a.i., using visual scales developed for each host species. The experiments were conducted in 2006 and repeated in 2007. Based on percent infection and disease severity, most V. dahliae isolates were highly aggressive on both host species. The tested isolates caused higher disease levels in the susceptible than in the moderately resistant phenotypes. They also caused more vascular discolouration in their original than in the alternative host. However, the isolates originating from sunflower caused less infection and disease severity on both hosts, compared to their potato counterparts. Cluster analysis based on all of the criteria used to assess pathogenicity led to three groups of isolates: (i) most V. dahliae potato isolates, which ranged with the highly aggressive control isolates, (ii) one V. dahliae sunflower isolate, which showed a similar pathogenicity level to the weakly-aggressive V. albo-atrum sub-group II control isolate, with no more symptoms than in the non-inoculated plants, and (iii) most V. dahliae sunflower isolates with mildly- to weakly-aggressive levels. Based on these results, V. dahliae cross-pathogenicity is very effective between potato and sunflower. Therefore, rotations involving these species should be avoided, especially where sunflower follows potato.     

<Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis> in the ornamental plant <Emphasis Type="Italic">Vinca minor</Emphasis> and its transmission through tomato seed

Two novel aspects of Tomato chlorotic dwarf viroid (TCDVd) are reported, namely that TCDVd was detected in symptomless plants of Vinca minor, a trailing ground cover surviving at subzero temperatures (−12°C); and that TCDVd was seed-borne in tomato and detected in high percentages in tomato seeds and seedlings. Soaking seeds in a low concentration of sodium hypochlorite did not eliminate the viroid. The sequence analysis showed that the TCDVd isolate consists of 360 nucleotides and has sequence identity between 96% to 99% with isolates of TCDVd from other hosts.     

Transmission of <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis> by bumblebees (<Emphasis Type="Italic">Bombus ignitus</Emphasis>) in tomato plants

Quantitative PCR revealed that Tomato chlorotic dwarf viroid (TCDVd) was present in substantial amounts in viroid-infected tomato flowers. Healthy tomato plants were arranged in two different glasshouses, and plants were mechanically inoculated with TCDVd. Bumblebees (Bombus ignitus) were then introduced into the glasshouses to reveal whether the viroid was transmitted from infected source plants to neighbouring healthy plants. TCDVd infection was found in neighbouring tomato plants more than 1 month after the introduction of the bees, some of which expressed symptoms, in both glasshouses. Thus, bumblebees transmitted TCDVd from tomato to tomato by pollination activities.     

Resistance to <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> expresses a hypersensitive-like response in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28’ was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28’ roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28’ when compared to the susceptible cultivar ‘IAC 15’. By contrast, ‘UFV 408-28’ was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15’ roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28’—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Seed and pollen transmission of <Emphasis Type="Italic">Apple latent spherical virus</Emphasis> in apple

To examine whether Apple latent spherical virus (ALSV) has spread among apple trees in an orchard, we surveyed 21 apple trees surrounding two ALSV-infected trees for virus infection using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). None of the 21 trees were infected, indicating that ALSV has not spread from the infected trees to the neighboring apple trees since it was first detected in 1984. We analyzed seed embryos and seedlings derived from infected trees and detected ALSV in 10 of 223 seed embryos (4.5%) and 10 of 227 seedlings (4.4%). From these results, we conclude that ALSV is seed-transmitted at a rate of ca. 4.5% in apple. We also analyzed seed embryos and seedlings from uninfected apple trees that were hand-pollinated with pollen from infected trees. We detected ALSV in only 1 of 260 seed embryos and in none of the 227 apple seedlings. This result indicated that the seed transmission rate via infected pollen is only 0–0.38%. In situ hybridization analysis of ALSV-infected apple flower buds showed that ALSV was present inside almost all pollen grains and in all ovary and ovule tissues, including the embryo sac and inner integument.     

Distribution of <Emphasis Type="Italic">Dickeya</Emphasis> spp. and <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> in naturally infected seed potatoes

Detailed studies were conducted on the distribution of Pectobacterium carotovorum subsp. carotovorum and Dickeya spp. in two potato seed lots of different cultivars harvested from blackleg-diseased crops. Composite samples of six different tuber sections (peel, stolon end, and peeled potato tissue 0.5, 1.0, 2.0 and 4.0 cm from the stolon end) were analysed by enrichment PCR, and CVP plating followed by colony PCR on the resulting cavity-forming bacteria. Seed lots were contaminated with Dickeya spp. and P. carotovorum subsp. carotovorum (Pcc), but not with P. atrosepticum. Dickeya spp. and Pcc were found at high concentrations in the stolon ends, whereas relatively low densities were found in the peel and in deeper located potato tissue. Rep-PCR, 16S rDNA sequence analysis and biochemical assays, grouped all the Dickeya spp. isolates from the two potato seed lots as biovar 3. The implications of the results for the control of Pectobacterium and Dickeya spp., and sampling strategies in relation to seed testing, are discussed.     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

<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.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

Feeding potential of <Emphasis Type="Italic">Cryptolaemus montrouzieri</Emphasis> against the mealybug <Emphasis Type="Italic">Phenacoccus solenopsis</Emphasis>

Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is an exotic species native to the USA, damaging cotton and other plant families. The feeding potential of different development stages of Cryptolaemus montrouzieri Mulsant, a biological control agent against mealybugs, was investigated on different development stages of P. solenopsis. Fourth instar grubs and adults of C. montrouzieri were the most voracious feeders on different instars of mealybug. The number of 1^st instar nymphs of mealybug consumed by 1^st, 2^nd, 3^rd and 4^th instar larvae and adult beetles of C. montrouzieri was 15.56, 41.01, 125.38, 162.69 and 1613.81, respectively. The respective numbers of 2^nd and 3^rd instar nymphs of mealybug consumed were 11.15 and 1.80, 26.35 and 6.36, 73.66 and 13.32, 76.04 and 21.16, 787.95 and 114.66. The corresponding figures for adult female mealybugs were 0.94, 3.23, 8.47, 12.71 and 73.40, respectively. The results indicate that C. montrouzieri has the potential to be exploited as a biocontrol agent in North India; inoculative releases of 4^th instar larvae and adults may provide instant control of P. solenopsis. Field experiments should be conducted to determine the efficiency of the ladybird on this mealybug.     

Effect of seed priming with <Emphasis Type="Italic">Serratia plymuthica</Emphasis> and <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> to control <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in different oilseed rape cultivars

The efficacy of a seed treatment of oilseed rape (OSR) (Brassica napus) with the rhizobacteria Serratia plymuthica (strain HRO-C48) and Pseudomonas chlororaphis (strain MA 342) applied alone or in combination against the blackleg disease caused by Leptosphaeria maculans was tested with different cultivars. Seeds were soaked in bacterial suspensions (bio-priming) to obtain log₁₀ 6–7 CFU seed⁻¹. Cotyledons were inoculated with a 10 ul droplet of L. maculans spore suspension of log₁₀ 7 spores ml⁻¹ and the disease index (size of lesions) was evaluated 14 days later. A mean disease reduction of 71.6% was recorded for S. plymuthica and of 54% for P. chlororaphis. The combined treatment was not superior to the treatment with S. plymuthica alone. The reduction of the disease caused by S. plymuthica was independent of the cultivar’s susceptibility, whereas the control effect recorded with P. chlororaphis increased with decreasing cultivar resistance to blackleg disease. The bacterial colonization of OSR was restricted to the roots and hypocotyl. No significant difference in bacterial colonization of the rhizosphere was observed between different cultivars, nor between single or combined bacterial seed treatments.     

Multifarious activity of bioformulated <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> PS1 and biocontrol of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Indian rapeseed (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.)

PGPR strain Pseudomonas fluorescens PS1 was evaluated to formulate carrier based bioformulations. The viability of P. fluorescens PS1 was monitored at different time intervals during the period of storage at room temperature in different carriers such as soil, charcoal, sawdust and sawdust-soil. Sawdust-soil was found to be the most efficient carrier material for P. fluorescens PS1 followed by other carriers. After 1 year of storage, P. fluorescens PS1 was re-isolated and assayed for its antifungal activity against Sclerotinia sclerotiorum a phytopathogenic fungus causing stem blight in Indian mustard, Brassica campestris. Results of scanning electron microscopy exhibited that P. fluorescens PS1 caused morphological alteration in mycelia of S. sclerotiorum as evident by hyphal perforation, and fragmented lysis. Seed bacterization of B. campestris with P. fluorescens PS1 induced enhanced seed germination, increased overall plant growth as well as reduced stem blight in mustard with improved yield. These findings demonstrate that P. fluorescens PS1 has significant potential to raise disease-free crops due to the presence of a wide array of PGP characteristics.