Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale)

Melon necrotic leaf spot virus (MNSV) caused a major outbreak of a leaf necrosis disease of hydroponically-grown cucumber plants at Humberside in 1983. The virus had c. 33 nm diam. particles which reacted serologically with MNSV antiserum of Dutch or American origin. Virus particles, which contained a single polypeptide (mol. wt 45 × 10³) and a presumed RNA species (mol. wt 1.5 × 10⁶), had a sedimentation coefficient (s_20.w) of 134 S and a buoyant density in caesium chloride of 1.35 g/cm³. The virus was mechanically transmissible, confined to species of Cucurbitaceae, transmitted by zoospores of Olpidium radicale and retained in the resting spores of the fungus. MNSV is thus both water-borne and soil-borne. O. radicale zoospores were killed in <5 min in suspensions containing 20 μg/ml of the surfactant Agral (alkyl phenol ethylene oxide). The disease did not reappear in 1984 when the cucumber crops were fed with nutrients containing 20μg/ml Agral.     

Application of plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus (PRSV-W) and Tomato chlorotic spot virus (TCSV) are responsible for severe losses in cucurbits and tomato production in south Florida and other regions in the USA. Traditional chemicals are not effective to control these viruses. Using plant growth-promoting rhizobacteria (PGPR) may present an alternative to control these viruses. Results from this study demonstrated that applying mixtures of PGPR strains is more efficient to control PRSV-W and TCSV compared to individual PGPR strain only. The application method significantly affected the efficiency of PGPR to control PRSV-W and TCSV. The highest reduction in disease severity of both PRSV-W and TCSV occurred in case of soil drenching with PGPR, followed by root dipping and seed coating treatments. Application of PGPR mixtures of (IN937a & SE34) or (IN937a &, SE34 & T4) were the most efficient methods to control these viral diseases.     

The regeneration and screening of watercress somaclones for resistance to Spongospora subterranea f. sp. nasturtii and measurement of somaclonal variation

A range of watercress (Rorippa nasturtium-aquaticum) explants (stems, hypocotyls, true-leaves, cotyledons and petioles) were tested for their capacity to regenerate adventitious shoots from callus formed using Murashige and Skoog medium containing different concentrations of thidiazuron and 2,4-dichlorophenoxyacetic acid. The highest shoot regeneration rate was a mean of 18 shoots per responding explant from stem callus formed on medium containing 5 μM thidiazuron and 0.05 μM 2,4-dichlorophenoxyacetic acid. A histological study confirmed that shoots originated directly from callus tissue. Twenty five percent of somaclones exhibited somaclonal variation in leaf shape, plant height, axillary branching or ploidy. The variation in 6% of somaclones was heritable to the first selfed generation. A screening protocol was developed to permit the identification of somaclones with increased resistance to the economically damaging watercress root pathogen, Spongospora subterranea f. sp. nasturtii. Although 883 somaclones were screened using this protocol, no significant increase in disease resistance was detected. This revised version was published online in June 2006 with corrections to the Cover Date.     

Integration of chitosan and plant growth-promoting rhizobacteria to control Papaya ringspot virus and Tomato chlorotic spot virus

Papaya ringspot virus-W (PRSV-W) and Tomato chlorotic spot virus (TCSV) are common viruses infecting vegetables in south Florida. Application of plant growth-promoting rhizobacteria (PGPR) has emerged as a potential alternative of chemical pesticides to control these viruses. But, it is not sufficient to completely replace chemical control. This study aimed to investigate the synergistic effect of chitosan and PGPR to control PRSV-W and TCSV. The efficiency of PGPR to suppress PRSV-W and TCSV was significantly improved when they were accompanied with chitosan treatment. The highest reduction in disease severity of both PRSV-W and TCSV was achieved when chitosan treatment was accompanied with mixture of two PGPR (IN937a + SE34) or three PGPR strains (IN937a + SE34 + SE56). The results of this study proved that implementation of chitosan and PGPR could significantly restrict losses due to PRSV-W and TCSV in squash and tomato, in Florida and the United States.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

In vitro culture of the obligate parasite Spongospora subterranea (cercozoa; plasmodiophorida) associated with root-inducing transferred-DNA transformed potato hairy roots

Spongospora subterranea is a soil-borne, obligate parasitic protist that causes powdery scab of potatoes. In this study, an in vitro culture system was developed for the maintenance and proliferation of the protist in potato hairy roots. The hairy roots of potato were induced in vitro with Agrobacterium rhizogenes. Cystosori of S. subterranea from potato scab lesions were surface disinfested and used to inoculate potato hairy roots. Plasmodia, zoosporangia, and cystosori were observed microscopically in the hairy roots within 6 wk after inoculation, indicating the completion of the life cycle of S. subterranea in vitro. This is the first in vitro culture system for S. subterranea, and will be a valuable tool to study fundamental and practical aspects of the biology of the parasite.     

Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis

Expression condition for maximum recovery of recombinant Apple chlorotic leaf spot virus (ACLSV) coat protein was standardized. The in vitro expressed fusion protein with 6xHis tag (~43 Kd) was purified from inclusion bodies and used as an antigen for raising polyclonal antiserum in rabbit. This antiserum consistently detected ACLSV in pome and stone fruits as well as herbaceous host plants by direct double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and direct tissue blot immunoassay (DTBIA). The conditions for immuno-capture RT-PCR (IC-RT-PCR) were also standardized.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Changes in the antioxidative metabolism induced by Apple chlorotic leaf spot virus infection in peach [Prunus persica (L.) Batsch]

Apple chlorotic leaf spot virus (ACLSV) is the causal agent of “viruela” disease, one of the limiting factors to apricot production in affected areas in the Southeast of Spain. In this work, the response of antioxidant enzymes to ACLSV infection of an indicator peach genotype, ‘GF305’, which is characterised by a great susceptibility to this virus, was studied before (short-period incubation) and after (long-period incubation) a cold treatment. Short-period ACLSV incubation caused significant changes in ascorbate peroxidase (APX), peroxidase (POX) and catalase (CAT) activities. In addition, long-period ACLSV incubation caused significant changes of activities in most of the antioxidant enzymes examined. The results show increases in the APX, dehydroascorbate reductase (DHAR), superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities, whereas POX suffered a decrease of about 34%.No changes in lipid peroxidation, measured as TBARS, were observed in peach leaves as a consequence of the long-period ACLSV incubation. Overall, the data show that long-period ACLSV incubation did not produce any symptoms in peach GF305 leaves or damage to membranes (no changes in lipid peroxidation), and this response was correlated with an increase in the antioxidant defences in leaves, such as the ASC-GSH cycle enzymes and the SOD and GST activities.     

The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh.

Potato mop-top virus (PMTV) was best detected in field soils by air-drying them for more than a week before remoistening and growing seedlings of Nicotiana tabacum or N. debneyi for a 6–10 week period. Infection of N. tabacum was assessed by inoculating sap from roots and shoots to Chenopodium amaranticolor. Similar inoculations from N. debneyi were far less convenient for detecting PMTV than recording leaf symptoms, but slightly more efficient. Air-dry soil retained PMTV infectivity for 9 months, when passed through a 50 μ sieve or when diluted with 10³ but not 10⁴ parts of steamed soil. Tobacco seedlings were not infected when their roots were steeped in PMTV-containing tobacco sap. Infective soils contained Spongospora subterranea, spore balls of which resisted air-drying for more than a year and passed a 50 μ sieve. Roots of susceptible seedlings were infected with PMTV when exposed to spore balls of S. subterranea taken from powdery scabs on PMTV-infected potato tubers, or to suspensions obtained by steeping, in nutrient solution, roots infected with virus-carrying cultures of S. subterranea. Plants in several families were hosts of S. subterranea, but probabilities of infection when exposed to spore balls differed greatly between families and only species of Solanaceae were good hosts. The ten species infected with PMTV when grown in infective soil or when exposed to spore balls of S. subterranea taken from PMTV-infected potato tubers are all members of this family. PMTV seems to be carried internally in S. subterranea spore balls and survived in them for at least a year. PMTV was transmitted by S. subterranea to Arran Pilot potato, causing yellow blotches in some leaves and spraing in many tubers. However, when newly infected with PMTV in the field, not all Arran Pilot tubers developed spraing. Also, although many spraing-affected or symptomless but PMTV-infected tubers carried PMTV-containing spore balls of S. subterranea, powdery scabs were rarely found near the centres of the rings of primary spraing. PMTV became established in virus-free soil when PMTV-infected tubers carrying S. subterranea were planted as ‘seed’ but not when virus-free tubers bearing powdery scabs were used. 5. subterranea seems the main, and possibly the only, vector of PMTV in the soils examined. S. subterranea did not transmit potato aucuba mosaic virus from potato to N. debneyi or Capsicum annuum.     

Studies on the seed-borne phases of dark leaf spot Alternaria brassicicola and grey leaf spot Alternaria brassicaeof brassicas

Tests in Britain on samples of basic and commercial Brassica oleracea seed between 1976 and 1978 showed that many lots were infected with Alternaria brassicicola. A. brassicae was uncommon in basic seed in these years and in commercial seed harvested in 1976 and 1977 but was frequent in seed harvested in 1978. Most affected seeds were contaminated by surface-borne spores and mycelium of A. brassicicola but many were internally infected by the fungus situated within the seed-coat and in some seeds in the embryo tissues. Superficial contamination by the fungus declined rapidly after 2 yr in cabbage seeds stored at 10 °C, 50% r.h. but internal infection persisted for up to 12 yr. In some samples, internal infection was commonly associated with small shrivelled seeds. Surface contaminated and internally infected seeds transmitted the disease but seedling infection was more closely correlated with the latter.     

The 50-kDa protein of Apple chlorotic leaf spot virus interferes with intracellular and intercellular targeting and tubule-inducing activity of the 39-kDa protein of Grapevine berry inner necrosis virus

To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.     

Calla lily chlorotic spot virus from Spider Lily (Hymenocallis litteralis) and Tobacco (Nicotiana tabacum) in the South‐west of China

Leaf samples were collected from four spider lily and tobacco plants with tospovirus‐like symptoms in Yunnan province. The nucleocapsid ORFs of the four isolates were obtained by RT‐PCR, cloning and sequencing of the amplified products. Nucleotide sequences of the obtained amplicons were more than 90% identical to that of Calla lily chlorotic spot virus (CCSV) isolates deposited in the GenBank database. The virus induced systematic leaf necrosis, chlorotic lesions, rugosity and deformation on mechanically inoculated experimental hosts. This is the first report of CCSV in mainland China, and also on spider lily and tobacco.     

Comparison of fungicides for control of leaf spot (Septoria apiicola) of celery

Of twenty-one fungicides tested at recommended rates of application for their effectiveness in controlling leaf spot of celery grown in Ireland on peat and on a clay-loam soil, three formulations containing fentin acetate with maneb and formulations of fentin hydroxide and fentin chloride were most effective. Benomyl was superior to the other non-tin fungicides tested and, at moderate disease levels, it resulted in marketable yields of celery equal to those recorded after treatment with the organotin compounds. Generally, the organotin compounds and benomyl were followed in descending order of effectiveness by copper oxychloride, captafol, Daconil 2787 and maneb. None of the fungicides tested in the three seasons caused visible symptoms of phytotoxicity and, on disease-free celery in 1969, none had a significant effect on yield.     

The role of pigmentation in survival of the leaf spot fungus Cercospora beticola

Both germination and survival time of spores of a pigmented strain of Cercospora beticola were directly correlated with relative humidity. Pigmented cultures, under conditions of low water availability (15%) survived 242 days' continuous exposure to light (110 lx). The ability of various strains of the fungus to survive in soil was found to be influenced by pigmentation, rate of growth, inoculum ratio in the soil and the physiological state of the organism before exposure to the soil environment.     

Studies on the taro leaf blight fungus Phytophthora colocasiae in Solomon Islands: control by fungicides and spacing

Surveys in 1974–75 in which 20 plants were examined in each of 105 fields showed that cabbage stem weevil was widespread on spring oilseed rape in the south of England, the larvae sometimes infesting a large percentage of plants and reducing vigour and yield. In replicated field trials during 1974–77, infestations were reduced by seed treatments of gamma-HCH or sprays of gamma-HCH, azinphos-methyl, azinphos-methyl + demeton-S-methyl sulphone, chlorpyrifos or triazophos. In some experiments treatments significantly improved plant growth or yield, or both. Granules of carbofuran or phorate also reduced larval infestations and damage, but both thiofanox granules and dimethoate sprays were ineffective. Sprays of gamma-HCH or azinphos-methyl + demeton-S-methyl sulphone which were effective against stem weevil could also in some years give improved control of blossom beetles (Meligethes spp.)     

Studies on the biology and control of cavity spot of carrots

Using a selective medium of corn meal agar with pimaricin and rifamycin, isolations from asymptomatic periderm of carrots grown on experimental plots or in commercial crops most frequently yielded the fast-growing species Pythium intermedium, P. sylvaticum or P. ultimum. In contrast isolations from cavity spot lesions mostly produced the slow-growing species P. violae and P. sulcatum. Following treatment of crops with metalaxyl + mancozeb, few isolations from asymptomatic periderm produced Pythium spp. and generally there was a reduction in the number of cavities. Treatment had little or no effect on size distribution of cavities. Cavity spot incidence was significantly less at higher pH values, fields of pH 8·0 and above producing carrots with little or no disease.     

空心莲子草叶斑病菌及代谢产物除草活性研究

从空心莲子草叶斑病叶或茎分离的镰刀菌及代谢产物对空心莲子草具有杀灭作用。代谢产物主要作用于空心莲子草叶片,使叶片组织变黄,最后枯萎死亡,发病死亡较快。用病菌分生孢子接种时致使植株叶片组织失水、发黄、脱落或从节间处折断枯死。开始时局部发病,有病斑形成,最后全株发病枯死。