<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

Somatic embryogenesis from anthers of the autochthonous <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Domina leads to <Emphasis Type="Italic">Arabis mosaic virus</Emphasis>-free plants

Attempts to conserve and utilise autochthonous grapevine germplasm in modern breeding programmes, are sometimes faced with the challenge that virus-free plants of old grapevine varieties and clones are hard to find. From 50 year-old vineyards in Frankonia the Vitis vinifera cv. Domina was selected showing particularly interesting loose-bunch architecture with fewer berries. However this valuable germplasm was carrying an Arabis mosaic virus (ArMV) infection requiring a reliable and effective method to produce healthy mother plants for clonal selection. Somatic embryogenesis was established from anthers as the most promising technical approach. The absence of ArMV in 46 regenerated plant lines was confirmed by ELISA and IC-RT PCR, repeated after different time intervals in vitro and in vivo after acclimatisation, and after one dormancy period under glasshouse conditions. Morphologically, all grapevines appeared true-to-type, and a screening of 20 plants by flow cytometry to determine the ploidy level and to exclude the risk of undesired genetic variability confirmed that all tested plants were diploid. Field evaluations of the initially selected bunch traits are currently underway.     

Detection and Elimination of Viruses in Callus, Somatic Embryos and Regenerated Plantlets of Grapevine

The distribution of some grapevine viruses in flower explants, embryogenic and non-embryogenic calli, single somatic embryos and plants regenerated from embryogenic cultures was investigated by RT-PCR and ELISA. Immature anthers and ovaries of the cultivars Grignolino infected by GRSPaV, GLRaV-1 and GVA, Müller-Thurgau infected by GRSPaV and GLRaV-3 and Bosco infected by GRSPaV were cultivated on media inducing indirect somatic embryogenesis. Viruses were detected both in anthers and ovaries. Four months after culture initiation 65.6% of tested calli were infected by at least one virus; high percentages of virus infection were found in calli originating from ovaries. No virus was detected in calli tested 8 months after culture initiation, as well as in single somatic embryos or in embryo-derived plantlets. Somatic embryogenesis confirmed its effectiveness in eliminating phloem-limited grapevine viruses. Regeneration of RT-PCR negative plantlets occurred even when at least a sector of the callus was still infected: the mechanism whereby somatic embryos are freed of some viruses could be related to the rapid proliferation of embryogenic cells within the callus or to the origin of the embryogenic callus from virus-free cells within the original explant.     

<Emphasis Type="Italic">In vitro</Emphasis> bacterization of banana (<Emphasis Type="Italic">Musa</Emphasis> spp.) with native endophytic and rhizospheric bacterial isolates: Novel ways to combat <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Compared to conventional planting material, micropropagated plantlets are highly susceptible to Fusarium wilt because they are free from beneficial root inhabitants. We aimed to introduce mixtures of beneficial microbes in the plantlets in the rooting medium under in vitro conditions rather than by field applications. Endophytes and rhizobacteria from different banana cultivars and plantation areas were screened and characterized. Under in vitro conditions, banana tissue culture plantlets were bacterized with the prospective endophytes, Bacillus subtilis strain EPB56 and EPB10 and the rhizobacteria, Pseudomonas fluorescens strain Pf1 and effects of in vitro bacterization were investigated against Fusarium oxysporum f. sp. cubense race 1 under glasshouse and field conditions. Inoculation of bananas during micropropagation allowed for the omission of minerals and salts as well as vitamins from the growing media while resulting in plantlets close to double size compared to the controls with full strength media. All endophyte and rhizobacteria strains tested resulted in significant reductions in Fusarium infection in the glasshouse and field and in significantly better plant growth. The three-way combination of bacteria resulted in 78% disease reduction and more than doubled the yields compared to the untreated controls across two field experiments. Three-way inoculation led to yields of 23 and 24 kg/ bunch compared to chemical disease control (13; 15 kg/bunch) and untreated controls (10; 13 kg/bunch) in the two field experiments. Under glasshouse conditions, activity of defence enzymes was significantly increased by all inoculation treatments. Inoculation in vitro led to the establishment of the microorganisms in the plant system before delivering to the farming community. Micropropagation combined with the establishment of a beneficial microbial consortium should complement the micropropagated plants for easier adaptation under field conditions.     

Epidemiology of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> on tomato

Leaf spot of tomato, incited by Pseudomonas syringae pv. syringae, has been reported recently in Italy on grafted and non-grafted tomato plants (scion Cuore di Bue, rootstock Solanum lycopersicum x Solanum hirsutum cv. Beaufort). In some greenhouses, more than 80% of plants were affected, with a marked reduction in yield. This work was undertaken in order to understand the effect of the number of hours of incubation at high relative humidity (r.h.) and temperature as well as the effect of the presence of wounds at infection time on the development of leaf spot. A difference in sensitivity to leaf spot was observed in the various cultivars tested, in terms of severity of P. syringae pv. syringae, with “Cuore di Bue” being the most susceptible of these cultivars. The development of leaf spot is mostly favored by the presence of wounds, at temperatures between 15 and 20°C. The severity of the disease is lower at 10 and 25°C and very low at 30°C. Under the most favorable temperature conditions, the presence of wounds is sufficient to allow the development of the pathogen immediately upon incubation at high r.h. The effect of wounds and the relatively low requirement of hours of incubation at high r.h. suggest the need for careful management and handling of plants when temperatures range between 15 and 25°C, and particularly within 15 and 20°C. All operations carried out, particularly at transplant and immediately after, should avoid the creation of wounds.     

A natural fungicide for the control of <Emphasis Type="Italic">Erysiphe betae</Emphasis> and <Emphasis Type="Italic">Erysiphe cichoracearum</Emphasis>

This study examines the effects of a vegetable fungicide on sugar beet powdery mildew (Erysiphe betae) and cucumber powdery mildew (Erysiphe cichoracearum). The formulations consisting of a dispersion of Brassicaceae meal in vegetable or mineral oils on infected leaves of sugar beet, reared in the greenhouse, and of musk melons cultivated under plastic tunnels, were tested in comparison to each oil taken separately. Both formulations containing Brassicaceae meals, caused 94% of conidia to be distorted while for the untreated group only 2% were distorted. Furthermore, the leaf area infected by E. betae was 56% for untreated plants and 2.7 and 9.9% respectively, for plants treated with meal containing mineral and vegetable oil. Vegetable oil considered separately or with Brassicaceae meals showed no phytotoxicity, while the formulations based on mineral oil showed a significantly lower fresh and dry weight on tomato plants. The low level or absence of phytotoxicity of plants treated with vegetable oil formulations suggests that to improve the efficacy of powdery mildew control, they could be used mixed with sulphur. The efficiency of the vegetable formulations in the powdery mildew control observed during these trials encourages further investigation on other parasitic fungi and foliar pathogens.     

Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.     

High incidence of <Emphasis Type="Italic">Pelargonium line pattern virus</Emphasis> infecting asymptomatic <Emphasis Type="Italic">Pelargonium</Emphasis> spp. in Spain

Geraniums (Pelargonium spp.) are traditional ornamental plants largely cultivated in Europe and northern America. Vegetative propagation makes them prone to viral infections, which have detrimental effects on crop production and quality. Asymptomatic samples collected in Spain were tested for a range of viruses using ELISA. The tobamovirus, Tobacco mosaic virus (TMV), the cucumovirus, Cucumber mosaic virus (CMV), and several viruses in the family Tombusviridae, namely, Pelargonium line pattern virus (PLPV), Pelargonium flower break virus (PFBV), and Pelargonium leaf curl virus (PLCV), were detected either singly or in combination in 59.2% of 800 samples. PLPV and PFBV infections were confirmed by dot-blot hybridisation. The most relevant viral infection found on Spanish asymptomatic geraniums was by Pelargonium line pattern virus (PLPV). Symptoms did not develop for 3 years on most of the PLPV infected geranium plants under greenhouse conditions.     

Epidemiology of root rot caused by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The infection of above-ground tissues of Brassica napus by Leptosphaeria maculans is well understood. However, root infection (root rot) under field conditions, the development of root rot over time and its relationship to other disease symptoms caused by L. maculans has not been described. A survey of B. napus crops was conducted in Australia to investigate the incidence and severity of root rot. Additionally, the pathway of root infection was examined in field experiments. Root rot was present in 95% of the 127 crops surveyed. The severity and incidence of root rot was significantly correlated with that of crown canker; however, the strength of this relationship was dependent on the season. Root rot symptoms appeared before flowering and increased in severity during flowering and at maturity, a pattern similar to crown canker suggesting that the infection of the root is an extension of the crown canker phase of the L. maculans lifecycle. All isolates of L. maculans tested in glasshouse experiments caused root rot and crown canker in B. napus and Brassica juncea. In the field, the main pathway of root infection is via invasion of cotyledons or leaves by airborne ascospores, rather than from inoculum in the soil. Root rot was present in crops in fields that had never been sown to B. napus previously, in plants grown in fumigated fields, and in glasshouse-grown plants inoculated in the hypocotyl with L. maculans.     

<Emphasis Type="Italic">Colletotrichum destructivum from</Emphasis> cowpea infecting <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and its identity to <Emphasis Type="Italic">C. higginsianum</Emphasis>

Colletotrichum isolates isolated from cowpea in the Hangzhou area of China were identified as C. destructivum based on morphological characteristics, pathogenicity tests, sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal DNA and the infection process. The ability of the C. destructivum isolates to infect Arabidopsis thaliana was investigated under laboratory conditions and showed a two-phase hemibiotrophic infection process. In addition, the sequences of the rDNA ITS region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. This article presents new evidence in support of C. higginsianum as a synonym of C. destructivum.     

Control of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> at a strawberry nursery by paddy-upland rotation

Effects of crop rotation between rice paddy fields and strawberry nurseries on the control of Verticillium wilt of strawberry were studied. For detecting Verticillium dahliae, the causal agent of Verticillium wilt, in soil, eggplant was used as an indicator plant. We were thus able to detect as low as 1 microsclerotium/g dry soil. In field surveys of Chiba and Hokkaido from 2000 to 2003, V. dahliae was detected in 9 of 10 upland fields but in none of 21 paddy-upland fields. In Hokkaido during 2000–2007, strawberry mother plants were planted, and plantlets were produced in upland and paddy-upland fields to assess V. dahliae infestation. Verticillium wilt of strawberry had never occurred in 72 tested paddy-upland fields, compared to 13.2–73.9% of plantlets infected with V. dahliae in upland fields. In a pot experiment in a greenhouse, two flooding treatments or two paddy rice cultivations suppressed Verticillium wilt symptoms on eggplant. In field experiments, one paddy rice cultivation in Chiba and two in Hokkaido prevented development of Verticillium wilt symptoms on eggplant. Verticillium wilt of strawberry was controlled completely with one paddy rice cultivation in infested fields in Chiba. In these field experiments, the number of microsclerotia of V. dahliae decreased under the flooding conditions for paddy rice cultivation. Based on the reduction in microsclerotia, a crop rotation system with paddy rice for 3 years (three times), green manure for 1 year, and strawberry nursery for 1 year was designed for Hokkaido.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Rapid screening of <Emphasis Type="Italic">Musa</Emphasis> species for resistance to Fusarium wilt in an <Emphasis Type="Italic">in vitro</Emphasis> bioassay

In order to accelerate breeding and selection for disease resistance to Fusarium wilt, it is important to develop bioassays which can differentiate between resistant and susceptible cultivars efficiently. Currently, the most commonly used early bioassay for screening Musa genotypes against Fusarium oxysporum f. sp. cubense (Foc) is a pot system, followed by a hydroponic system. This paper investigated the utility of in vitro inoculation of rooted banana plantlets grown on modified medium as a reliable and rapid bioassay for resistance to Foc. Using a scale of 0 to 6 for disease severity measurement, the mean final disease severities of cultivars expressing different levels of disease reaction were significantly different (P ≤ 0.05). Twenty-four days after inoculation with Foc tropical race 4 at 10⁶ conidia ml⁻¹, the plantlets of two susceptible cultivars had higher final disease severities than that of four resistant cultivars. Compared with ‘Guangfen No.1’, ‘Brazil Xiangjiao’ is highly susceptible to tropical race 4 and its mean final disease severity was the highest (5.27). The plantlets of moderately resistant cultivar ‘Formosana’ had a mean final disease severity (3.53) lower than that of ‘Guangfen No.1’ (4.33) but higher than that of resistant cultivars: ‘Nongke No.1’, GCTCV-119, and ‘Dongguan Dajiao’ (1.87, 1.73, and1.53, respectively). Promising resistant clones acquired through non-conventional breeding techniques such as in vitro selection, genetic transformation, and protoplast fusion could be screened by the in vitro bioassay directly. Since there is no acclimatization stage for plantlets used in the bioassay, it helps to improve banana breeding efficiency.     

<Emphasis Type="Italic">Odontoglossum ringspot virus</Emphasis> causing flower crinkle in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3–100% nucleotide identity and 96.2–100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids.     

Reaction of <Emphasis Type="Italic">Leersia</Emphasis> grasses to <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> collected from Japan and Asian countries

The present study was conducted to determine if there is specificity in the host-pathogen relationship between the isolates of Xanthomonas oryzae pv. oryzae, the causal bacterium for rice blight and Leersia grasses, the alternative weed hosts of the disease. Plants of three species of Leersia, namely, L. sayanuka, L. oryzoides and L. japonica, were collected from various parts of Japan and were inoculated with the X. oryzae pv. oryzae isolates obtained from various locations in Japan and from 11 Asian countries. Four L. sayanuka plants were found susceptible to all Race II isolates and some Race I isolates, but were resistant to all Race III isolates. Race III is known to have a wider range pathogenicity to rice cultivar groups compared with Race I and II. Although the reactions of two L. oryzoides plants to Race I and II isolates were similar to that of L. sayanuka, the L. oryzoides plant collected from Niigata Prefecture showed a susceptible reaction to some Race III isolates. On the other hand, L. japonica plants gave reactions different those of L. sayanuka and L. oryzoides, with two plants of L. japonica found to be resistant to all test isolates collected from Japan. The Asian isolates exhibited a wide host range against the international differential rice cultivars, but almost all of them were avirulent to Leersia plants. These results indicate that the relationship between the pathogenicity of the causal bacterium and the resistance of host plants is very complex, and suggest that pathogenic diversity of X. oryzae pv. oryzae might be related to the resistance of Leersia spp.     

Inheritance of resistance in <Emphasis Type="Italic">Solanum nigrum</Emphasis> to <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

Solanum nigrum, black nightshade, is a wild non-tuber bearing hexaploid species with a high level of resistance to Phytophthora infestans (Colon et al. 1993), the causal agent of potato late blight, the most devastating disease in potato production. However, the genetic mode of resistance in S. nigrum is still poorly understood. In the present study, two S. nigrum accessions, 984750019 (N19) and #13, resistant (R) and susceptible (S), respectively, to three different isolates of P. infestans, were sexually crossed. The various kinds of progeny including F1, F2, F3, and backcross populations (BC₁; F1 × S), as well as two populations produced by self-pollinating the R parent and S parent, were each screened for susceptibility to P. infestans isolate MP 324 using detached leaf assays. Fifty seedling plant individuals of the F1 progeny were each resistant to this specific isolate, similarly to the seedling plants resulting from self-pollination of the resistant R parent. Thirty seedling plants obtained from self-pollination of the S parent were susceptible. Among a total of 180 F2 plants, the segregation ratio between resistant and susceptible plants was approximately 3: 1. Among the 66 seedling plants of the BC₁ progeny originating from crossing an F1 plant with the susceptible S parent, there were 26 susceptible and 40 resistant plants to P. infestans. The segregation patterns obtained indicated monogenic dominant inheritance of resistance to P. infestans isolate MP 324 in S. nigrum acc. 984750019. This gene, conferring resistance to P. infestans, may be useful for the transformation of potato cultivars susceptible to late blight.     

Seed transmission of<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">lactucae</Emphasis>

Twenty-seven seed samples belonging to the lettuce cultivars most frequently grown in Lombardy (northwestern Italy), in an area severely affected by Fusarium wilt of lettuce, were assayed for the presence ofFusarium oxysporum on a Fusarium-selective medium. Isolations were carried out on subsamples of seeds (500 to 1500) belonging to the same seed lots used for sowing, and either unwashed or disinfected in 1% sodium hypochloride. The pathogenicity of the isolates ofF. oxysporum obtained was tested in four trials carried out on lettuce cultivars of the butterhead type, very susceptible to Fusarium wilt. Nine of the 27 samples of seeds obtained from commercial seed lots used for sowing in fields affected by Fusarium wilt were contaminated byF. oxysporum. Among the 16 isolates ofF. oxysporum obtained, only one was isolated from disinfected seeds. Three of the isolates were pathogenic on the tested cultivars of lettuce, exhibiting a level of pathogenicity similar to that of the isolates ofF. oxysporum f.sp.lactucae obtained from infected wilted plants in Italy, USA and Taiwan, used as comparison. The results obtained indicate that lettuce seeds are a potential source of inoculum for Fusarium wilt of lettuce. The possibility of isolatingF. oxysporum f.sp.lactucae, although from a low percent of seeds, supports the hypothesis that the rapid spread of Fusarium wilt of lettuce observed recently in Italy is due to the use of infected propagation material. Measures for prevention and control of the disease are discussed. http://www.phytoparasitica.org posting Dec. 16, 2003.     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.