The Role of Infective Plant Debris, and its Concentration in Soil, in the Ecology of Tomato Mosaic Tobamovirus—a Non-vectored Plant Virus

Only a few plants in a crop are generally thought to become infected by abiotic soil transmission. In glasshouse experiments we have induced almost total infection in tomatoes growing in soil with infective debris, but levels of infection are dependent upon certain conditions. We found that as the inoculum concentration decreased (i) a greater percentage of the infections were either latent (symptomless) or restricted to roots and (ii) infection levels decreased. Thus, apparent infection (based on symptoms) of 0. 10% was in reality 0 60–70% based on enzyme-linked immunosorbent assay and immunoelectron microscopy testing of roots and leaves. This occurred whether seedlings or seed were planted into infected mix. Under conditions in which minimal root damage was caused (planting seed) or roots were mechanically inoculated only once, almost all systemically infected plants were symptomless.     

Systemic spread of downy mildew in basil plants and detection of the pathogen in seed and plant samples

Downy mildew on sweet basil (Ocimum basilicum L.) occurs worldwide. Contaminated seeds are considered as the primary inoculum source. So far no strategy to control the disease is available. Hence, the use of pathogen-free seeds is the only alternative to prevent disease outbreaks. Therefore, a rapid diagnostic method for seed testing is urgently needed. The sensitivity of a specific PCR method for direct detection of the downy mildew pathogen Peronospora belbahrii on basil samples, particularly on seeds, was evaluated. The applied PCR method proved to be very sensitive for direct detection of the pathogen on seeds and plant samples. The PCR detection limit of P. belbahrii in artificially infested seeds corresponded to the DNA amount of a single spore per seed. Additionally, the systemic spread of the pathogen from naturally infected seeds was investigated. The experiments showed that outgrowing basil plants were latently infected with the downy mildew pathogen, and the infection continued within the plant. Contaminated seeds were harvested from symptomless latently infected plants. These results support the implementation of PCR-based detection in a seed certification scheme and the necessity to control the pathogen on seeds. The PCR method can also be used for evaluation of pathogen control on seeds based on detection of the pathogen in outgrowing plants.     

Neck rot (Botrytis allii) of bulb onions

Experiments on neck rot of onions, caused by Botrytis allii showed that, although the disease only became evident in store, a major source of the pathogen was samples of infected seeds. In 1972 and 1973, 39·5 and 71·4% respectively of commercial onion seed samples tested at Wellesbourne were infected. The pathogen was internal in seed and persisted for 3 ½ yr in infected seeds kept in a seed store at 10°C and 50% r.h. Seedlings raised from diseased seeds became infected by mycelial invasion of the cotyledon leaf tips from seed-coats many of which remained attached to the cotyledons when seedlings emerged from the soil. The fungus attacked the living tissues of these leaves symptomlessly, producing conidiophores only after the leaf tissue senesced and became necrotic. Because the fungus was symptomless, the rate of spread of the pathogen in onion crops was assessed by incubating successive samples of plants from the field in humid conditions when infected tissues developed conidiophores of the fungus. This method showed that the disease was progressive in onion crops spreading more rapidly in wet humid conditions (e.g. 1972) than in dry ones (e.g. 1973). The principal means of spread were probably fungal spores; conidiophores bearing spores being produced abundantly on plants in the field under high humidity. The fungus invaded the leaves of plants successively, first infecting each leaf at the tip and then growing downwards in the tissues and eventually invading the neck of the onion bulb via the leaves which emerged directly from the top of the neck. By harvest, the fungus was situated deep within the neck tissues of infected maturing onion bulbs.     

Further studies on seed transmission of broad bean stain virus and Echtes Ackerbohnenmosaik -Virus in field beans (Vicia faba)

Broad bean stain virus (BBSV) and Echtes Ackerbohnenmosaik-Virus (EAMV) were detected in the seed coat and embryo sac fluid of immature seeds from infected field beans (Viciafaba minor) by inoculation to Phaseolus vulgaris; BBSV was also detected in immature embryos. The proportion of seeds infected with either virus decreased during maturation. The viruses were transmitted to seedlings as often through fully ripened seeds from which the seed coats had been removed as through intact seeds. Both viruses were detected in pollen from infected plants, but in glasshouse tests only BBSV was transmitted through pollen to seeds. Delaying fertilization in plants infected with BBSV or EAMV seemed not to affect seed transmission of either virus. In glasshouse tests BBSV was transmitted more often through seeds from plants that were inoculated before flowering than during flowering, and was not transmitted through seeds from plants inoculated after flowering; EAMV was transmitted only through seeds from plants inoculated before flowering. In tests on seed from naturally infected plants BBSV was transmitted more often through seeds from plants that developed symptoms before flowering than during flowering. Both viruses were seed-borne in all cultivars tested and there was no marked difference in the frequency of transmission of either virus among the spring-sown cultivars most common in Britain. Both viruses persisted in seed for more than 4 yr.     

Location of Soybean Mosaic Virus in Seed-Parts of Individual Dormant and Germinating Mottled and Non-mottled Soybean Seeds

Soybean mosaic virus (SMV) was detected in individual embryos of both dormant mottled (50%) and non-mottled (50%) seeds of certified soybean cvs Davis. Essex. Hutchinson, Ransom, Stonewall, and Young by using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). SMV was not detected in individual strained seed coats of mottled or non-stained seed coats of non-mottled dormant seeds and endosperms of either type of seed. SMV was not detected m individual stained and nonstained seed coats, epicotyls, and endosperms of mottled and non-mottled seeds at 72 h post-germination. However, the virus was detected in individual hypocotyls at an average level of 31% in post-germinated seeds (mottled and non-mottled) of all cultivars. Symptomless glasshouse-grown 6–8-week-old seedlings from mottled and non-mottled seeds of certified soybean cultivars occurred twice as often as those showing early symptoms. However, almost half of these symptomless plants were found to be SMV-infected by DASELISA. Virus-free soybean plants grown to maturity from non-mottled seeds also produced mottled seeds.     

Effect of Source Strength, Distance and Direction on the Spread of Maize Dwarf Mosaic Virus

Transmission of Pseudomonas avenae from rice seed to seedling and from plant to seed was shown. Based on experiments with unhulled and hulled seeds and on histopathological studies the location of the pathogen in seeds and the possible pathway of the pathogen from seed to seedling is suggested. Infected seeds were harvested from plants on which no symptoms were observed after the seedling stage. The experimental conditions indicated that the bacterium can be transmitted internally from plantto seed in latently infected plants.     

Transmission of seed-borne infection of chilli by Burkholderia solanacearum and effect of biological seed treatment on disease incidence

Abstract

A survey of chilli fields in the state of Karnataka, India, showed the presence of bacterial wilt disease in important chilli growing regions. The disease incidence ranged from 26?–?32%. The pathogen was isolated from infected plant material and seeds. Infected plant material showed the release of milky white bacterial ooze. Burkholderia solanacearum was detected from chilli seeds by liquid assay and its identity was confirmed by biochemical tests, hypersensitive reaction and pathogenicity tests. Seed transmission of the pathogen up to 45% was observed in seeds artificially infested with the pathogen. Among different tissues of the seed, endosperm showed the presence of the pathogen. Biological seed treatment with antagonistic Pseudomonas fluorescens significantly (p?=?0.05) improved the seed quality parameters under laboratory conditions and drastically reduced the bacterial wilt incidence under field conditions. Seed-borne nature, transmission and effect of Pseudomonas fluorescens in both the forms of pure culture and formulation on seed quality parameters and bacterial wilt incidence are discussed in the present work.     

The quantitative estimation of the infection of bean seed with Pseudomonas phaseolicola (Burkh.) Dowson

A routine laboratory method for the detection of Pseudomonas phaseolicola in bean seed is described. The method will detect low levels of seed-borne infection and has been used in a statistical procedure (the most probable number method) to give an estimate of percentage infection. Infection in seeds harvested from heavily infected crops varied from 10 to 1%, compared with from 1% to less than 0.1% in commercial seed stocks. A high proportion of infected seeds failed to produce infected plants and this may account for the very low levels of primary infection reported in the field. Removal of seeds showing possible ‘symptoms’ of disease reduced, but did not eliminate, infection from seed stocks.     

THE PATTERN OF FIELD INFECTION OF POTATO BY THE BEET RINGSPOT STRAIN OF TOMATO BLACK-RING, A SOIL-BORNE VIRUS

The potential importance of the beet ringspot strain of tomato black-ring, a soil-borne virus, was assessed by growing stocks of Kerr's Pink potato for 1 year on infested land and subsequently on uninfested land. The incidence of infection in two stocks was 39 and 8% in the first year on uninfested land, and 29 and 5% after 2 years.
The virus was usually restricted to the roots of plants in the first year of infection, but a few plants showed black rings and spots in their leaves. In the second year, 20–55% of the plants grown from tubers set by symptomless, but infected, mother plants were infected: many of these showed leaf necrosis, others had stunted shoots, and cupped and distorted leaves; some were symptomless although systemically infected. In the third and fourth years, most of the progeny from plants which had symptoms or which were symptomless but systemically infected, contained the virus: nearly all such infected plants were stunted and distorted or were symptomless. Infection decreased the weight of tubers produced by plants with severe necrotic spotting but not the yield of plants with less necrosis. The number and weight of tubers per plant were decreased by 15 and 20% respectively, in symptomless systemically infected plants, and by 20 and 30% in stunted plants.     

Detection of infection by Pseudomonas phaseolicola (Burkh.) Dowson in white-seeded dwarf bean seed stocks

Seeds of white-seeded varieties of dwarf beans infected with Pseudomonas phaseolicola may show fluorescent areas in the testa under ultra-violet light. Fluorescence is not visible in seed with dark-coloured testas and is masked by coloured seed dressings. Bacterial infection is not the only cause of fluorescence in testas and the type and colour of the fluorescence cannot be used to diagnose infection. Of 542 fluorescing seeds examined 112 were infected by the pathogen, and only six infected seeds were found in 495 non-fluorescing seeds from the same samples. It is estimated that in two seed stocks used for these experiments 64 and 68 % of infected seeds were of the fluorescent type. A 34 lb sample of seedstock has to be taken for examination to detect, with 95 % confidence, ten infected seeds in 1 cwt. The selection of all fluorescing seeds from such large samples effectively concentrates the infected seed into a sample small enough to be tested by bacteriological methods. Test seeds are examined bacteriologically by enrichment-culture in nutrient broth. Ps. phaseolicola is identified in these cultures from the diagnostic symptoms of halo-blight disease induced in bean seedlings inoculated with aliquots of the cultures.     

Detection of Xanthomonas oryzae pv. oryzae in artificially inoculated and naturally infected rice seeds and plants by molecular techniques

A polymerase chain reaction (PCR) technique was developed for detecting the presence of Xanthomonas oryzae pv. oryzae, the bacterial leaf blight (BLB) pathogen in rice seed and for studying the transmission of this bacterium from seed to plant. Primers TXT and TXT4R from an insertion sequence (IS1113) of the pathogen were used to amplify a 964-bp DNA fragment. A combined biological and enzymatic amplification (BIO-PCR) technique was used to detect the pathogen in naturally infected seed. The level of detection of TXT and TXT4R primers was 55 fg DNA of X. o. pv. oryzae, which is roughly the equivalent of seven cells (and four cells in pure culture suspension) of X. o. pv. oryzae. Hybridization of IS1113 with the amplified DNA fragment in Southern blot analysis confirmed that the 964-bp DNA fragment was amplified from X. o. pv. oryzae. The presence of the IS1113 element in strains of X. o. pv. oryzae from 16 rice-growing countries was confirmed by DNA dot blot analysis. X. o. pv. oryzae was detected from the seed washes and DNA extracted from the seed washes of naturally infected seeds of cvs Jaya and TN1. When stored at 4 degrees C, the pathogen was recovered up to 4 months and 9 months from naturally infected seeds of cvs Jaya and TN1, respectively. The BLB bacterium was also detected in seedlings, mature plants and seeds collected from plants raised from naturally infected seeds.     

Necrosis in field beans (Vicia faba) induced by interactions between bean leaf roll, pea early-browning and pea enation mosaic viruses

Mixed infections with two or three viruses - bean leaf roll (BLRV), pea early-browning (PEBV) and pea enation mosaic (PEMV) - were detected in plants showing leaf curling, stunting and necrosis in a crop of field beans grown for seed in 1980. In glasshouse tests, field bean plants infected with any one of these viruses showed no necrosis, and plants infected with PEBV and PEMV together showed symptoms of PEMV only. However, mixed infection with BLRV and PEMV almost invariably induced severe stunting and leaf necrosis, and infection with BLRV and PEBV often induced both leaf and stem necrosis and sometimes caused early death. Thus it seems that the necrotic symptoms seen in the field were induced by interactions between BLRV and the other viruses. No transmission of PEBV was detected through seed harvested from the crop, but up to 5% transmission was detected through seed from experimentally-infected plants. The infected seedlings were symptomless.     

The Probability of Detecting Clavibacter michiganensis subsp. sepedonicus by Indexing Seed Potato Lots with Serological Tests

Detection of the bacterial ring pot pathogen ( Clavibacter michiganensis subsp. spedonicus ) in seed potato lots by laboratory indexing complements visual inspection. The probability of detecting symptomless infections is a function of sample size and incidence of infection. We determined the incidence of asymptomatic stem and tuber infections in four potato cultivars at three levels of inoculum. At the high inoculum level, 51–93% of stems were infected at 80 days after planting, and 10–59% of the tubers were infected at harvest. The effect of the different percentages of infected stems and tubers on the probability of detection for simple random sampling was calculated for a constant sample size. The actual detection levels for two cultivars planted in field plots with predetermined incidence levels of ring rot infected plants were reasonably close to predicted probabilities.     

Transmission of Three Viroids Through Seed and Pollen of Tomato Plants

The severe strain of potato spindle tuber viroid (s-PSTV) as well as chrysanthemum stunt (CSV) and cucumber pale fruit (CPFV) viroids were found to be transmitted through seed and pollen of the tomato cvs. Rutgers and Najwcze?niejszy. Plants pollinated with a pollen infected with any of these three viroids became systematically infected. Plant, fruit and seed symptoms of viroid infection were noted on sap- and pollen-inoculated plants and the yield of these plants was reduced. Tomato cv. Rutgers plants grown from infected seeds were symptomless although all three viroids were detected in these plants by bioassay and by electrophoresis on 5% polyacrylamide gel. When DNA complementary to s-PSTV RNA was used for a direct viroid detection in seed samples by spot hybridization technique it hybridized not only with s-PSTV RNA but also with CSV RNA as well as with CPFV RNA.     

Incidence of potato virus Y infection in seed and ware tubers of the potato cv. Record

The incidence of potato virus Y (PVY) infection was assessed in samples of potato tubers, cv. Record, taken from Scottish seed stocks and English ware crops grown from some of these seed stocks. PVY was readily detected by ELISA of tuber sprouts. PVY-infected tubers were found in 10 seed stocks of 84 tested. The mean level of virus infection was 0.23%, 0.76% and 0.56% in Super Elite, Elite and AA stocks respectively. In 46 commercial ware crops grown from some of these seed stocks, a substantial proportion of the harvested tubers in all but one of the crops were infected with PVY, the mean percentage of infected tubers was 58.5%. Ware crops grown from seven seed stocks in which PVY had been detected (mean 6.2% infection in seed) contained a mean of 70% infected tubers, compared with 56% infection in crops grown from 39 stocks in which PVY was not detected in the seed tubers. The predominant PVY strain detected in the ware crops was the veinal necrosis strain (PVY^vn).     

Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

In Western Australia, infection with cucumber mosaic virus (CMV) was widespread in all three subspecies of subterranean clover (Trifolium subterraneum) growing in plots belonging to the Australian National Subterranean Clover Improvement Programme. Seed-borne CMV was detected in seed harvested in 1984–1986 of 18/25 cultivars from two collections of registered cultivars; seed transmission rates ranged up to 8.8%. Seed samples from CMV-inoculated plants of 11 cultivars transmitted the virus to 0.5–8.7% of seedlings. Seed transmission rates greater than 5% were obtained only with cvs Enfield, Green Range and Nangeela. CMV was not detected in seed harvested in 1975–1981 from one of the registered cultivar collections, in 17 commercial seed stocks from 1986 or in a survey of subterranean clover pastures.
Symptoms in subterranean clover naturally infected with CMV included mottle, leaflet downcurling and dwarfing but severity varied with cultivar and selection. CMV isolates from different sources varied in virulence when inoculated to subterranean clover; two (both from subterranean clover) were severe, two moderate and three (including one from subterranean clover) mild. In pot tests, CMV decreased herbage production and root growth (dry wts) of cv. Green Range by 49% and 59% respectively. In spaced-plants growing in plots, CMV decreased herbage production and root growth of cvs Green Range and Northam by 59–630 and seed production of cv. Green Range by 45%. In rows sown with infected seed, aphid spread increased infection levels to 75% in cv. Green Range and 44% in cv. Esperance and losses in herbage production of 42% and 29% respectively were recorded.
CMV isolated from subterranean clover included isolates from both serogroups.     

Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation

The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic separation (IMS), based on the use of immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10(6)IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10(3) to 10(0)CFU/ml. Threshold detection was lower than 10CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.     

The effect of iprodione on the seed-borne phase of Alternaria brassicicola

Alternaria brassicicola infection of Brassica oleracea seeds was effectively controlled by a dust application of iprodione (Rovral 50% w.P.). At 2.5 g a.i./kg the seed-borne fungus was usually eliminated from samples with up to 61.5% affected seeds (35.5% internally diseased) but higher levels of infection required increased doses for complete eradication of the fungus. The germination of healthy seeds, including samples from 7–yr-old stocks, on filter paper was unaffected by the treatment. However, the germination of diseased samples, particularly those internally infected with A. brassicicola, was improved. More seedlings emerged from iprodione treated than from untreated seeds in glasshouse soil but the differences were not significant. The application of gamma-hexachlorocyclohexane to iprodione treated seeds sown in soil did not adversely affect subsequent emergence or disease control. Disease control was maintained and germination was not affected by the treatment when treated infected seeds were stored for 2 yr at 10 °C, 50% r.h. In a field trial iprodione seed treatment reduced seedling infection in a cabbage crop grown from naturally diseased seeds (100% contaminated, 45.5% internally infected) from 5.6 to 0.04%.     

Studies on the survival and transmission of Xanthomonas manihotis on cassava seed

Cassava seed which had been stored at 5 ^oC and 60% r.h. for 2–51 months was assayed for the presence of Xanthomonas manihotis by a leaf-infiltration technique, using as inoculum the supernatant from seeds soaked in sterile water at 30 ^oC for 2–4 h. The threshold of sensitivity of the assay method was 10⁵ cells/ml. Twenty out of 50 samples yielded the pathogen. The infested seed had been in storage for 2–18 months. Bacteria reisolated from infiltrated leaves were identical to X. manihotis in cultural characteristics, phage type and pathogenicity. Surface sterilisation or hot air treatment for 24 h at 65^oC or lower did not eliminate the pathogen from infested seed. Soaking of infested seed in hot water at 60 ^oC for 20 min reduced the number of bacteria to less than the minimum detectable level without appreciably reducing germination. Cassava bacterial blight was observed in 8-wk-old seedlings which had been planted during the dry season at a site where infection from outside sources was unlikely. It is postulated that a low percentage of successful seed transmissions of X. manihotis can occur under favourable environmental conditions.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.