The Relationship Between Wheat Seed Weight, Infection by Fusarium culmorum or Microdochium nivale, Germination and Seedling Disease

The distribution of seeds by weight for three lots of winter wheat cv. Avalon infected by Fusarium culmorum and three lots of winter wheat cv. Riband infected by Microdochium nivale was determined. The distribution of infected seeds within each seed lot was then determined by isolating F. culmorum from seeds on moist filter paper and M. nivale from seeds on potato dextrose agar. The distribution of M. nivale infected seeds between seeds of different weight was similar to that of the seed lot as a whole, whereas the distribution of F. culmorum was greater in light seeds than heavy seeds. The percentage germination of infected seeds decreased with seed weight. A similar situation was found with respect to seedling emergence in compost for F. culmorum infected seeds. However, with M. nivale infection, similar numbers of seedlings emerged from both light and heavy infected seeds. Seed treatment with guazatine increased seedling emergence for both light and heavy seed infected by M. nivale. However, seedling emergence from F. culmorum infected seed was poor even following treatment with guazatine. Poor emergence was most evident from light seed.     

Seed transmission of maize downy mildew (Peronosclerospora sorghi) in Nigeria

In an area of Nigeria where downy mildew of maize is present, histological assessment of maize seed revealed the presence of mycelium and oospores of Peronosclerospora sorghi in the kernels. Seed transmission of downy mildew of maize was demonstrated when grain purchased at local markets gave mean seedling infection rates of 12·3% (untreated seeds) and 10·0% (in metalaxyl-treated seeds) within 7 days of emergence, after storage in a desiccator for 30 days. When untreated seeds taken from nubbin ears of systemically infected plants from four states in southern Nigeria were planted at 9 days (17–22% moisture content) and 27 days (9–22% moisture content) after harvest, 20·0% infected seedlings resulted in both trials. Seeds from Borno state in northern Nigeria had 26·6% systemic seedling infection after 9 months of storage at 11% moisture content. When seeds harvested from maize plants inoculated with P. sorghi through silks were examined histologically, hyphae of P. sorghi were observed mostly in the scutellum of the embryo. Transmission of disease to seedlings was observed when the silk-inoculated seeds (9% moisture content) were planted in pots in a greenhouse; however, no disease transmission was observed when such seeds were planted in the field. The epidemiological significance of seed transmission is discussed with particular reference to survival of inoculum and development of epidemics. Also noteworthy is the overall significance of seed transmission in Nigeria, where the major source of seed is that saved by farmers from their grain crop, occasionally supplemented by seed bought from the local market.     

苹果锈果类病毒在八棱海棠种子中的分布及氢氧化钠脱毒效果分析

为明确苹果锈果类病毒在八棱海棠果实和种子中的分布特征、种传率以及药剂脱除效果,以带毒母株上的八棱海棠果实和种子为试材,运用RT-PCR技术分析了八棱海棠果实不同部位锈果类病毒的带毒率、实生后代的带毒情况以及氢氧化钠脱除病毒的效果。结果表明,八棱海棠果皮、果肉、种子以及种胚均不同程度携带苹果锈果类病毒,其带毒率分别为96.0%、96.0%、52.0%和4.0%;该病毒可经种子传递给后代,种传率为12.1%;经2%氢氧化钠溶液浸种10、15、20 min,种子的病毒检出率均为0,但后代实生苗的带毒率分别为2.5%、1.3%和0。表明苹果锈果类病毒可侵染种子不同部位并经种子传递给后代,氢氧化钠浸种是脱除该病毒的有效方法。     

Tracing seed to seedling transmission of the wheat blast pathogen Magnaporthe oryzae pathotype Triticum

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT), initially restricted to South America, is a global threat for wheat after spreading to Asia in 2016 by the introduction of contaminated seeds, raising the question about transmission of the pathogen from seeds to seedlings, a process so far not well understood. We therefore studied the relationship between seed infection and disease symptoms on seedlings and adult plants. To accomplish this objective, we inoculated spikes of wheat cv. Apogee with a transgenic isolate (MoT-DsRed, with the addition of being resistant to hygromycin). We identified MoT-DsRed in experiments using hygromycin resistance for selection or by observation of DsRed fluorescence. The seeds from infected plants looked either apparently healthy or shrivelled. To evaluate the transmission, two experimental designs were chosen (blotter test and greenhouse) and MoT-DsRed was recovered from both. This revealed that MoT is able to colonize wheat seedlings from infected seeds under the ground. The favourable conditions of temperature and humidity allowed a high recovery rate of MoT from wheat shoots when grown in artificial media. Around 42 days after germination of infected seeds, MoT-DsRed could not be reisolated, indicating that fungal progression, at this time point, did not proceed systemically/endophytically. We hypothesize that spike infection might occur via spore dispersal from infected leaves rather than within the plant. Because MoT-DsRed was not only successfully reisolated from seed coats and germinating seeds with symptoms, but also from apparently healthy seeds, urgent attention is needed to minimize the risks of inadvertent dispersal of inoculum.     

携带GFP的MNSV侵染性克隆载体的构建

正甜瓜坏死斑点病毒(Melon necrotic spot virus,MSNV)属于番茄丛矮病毒科(Tombusviridae)香石竹斑驳病毒属(Carmovirus),其主要寄主是葫芦科作物。MNSV首次在我国江苏海门发生[1],随后在山东寿光[2]、青州市[3]及广西南宁[4]等地均有报道。种子带毒是该病害初侵染的主要来源[5],随着种子调运、设施栽培推广和甜瓜产业的发展,该病害日趋严重。MNSV侵染初期叶片、叶柄和茎杆出现坏死斑或     

携带GFP的MNSV侵染性克隆载体的构建

Melon necrotic spot virus (MNSV) is a newly reported melon-infecting virus species in China, which is seriously terrible in melon production. The MNSV full-length cDNA of MNSV was obtained by RT-PCR and was ligated to pXT1 vector to obtain the infectious clone vector pXT1-MNSV. The melon was inoculated with pXT1-MNSV, and melon leaves infected with pXT1-MNSV were collected and inoculated into the healthy melon by sap. The results showed that the infectious clone pXT1-MNSV was well pathogenic and could be transmitted by sap, so the Koch′s law was completed. Using MNSV infectious clone, the MNSV infectious clone vector pMNSV-cp-GFP harboring full-length GFP was constructed, Because GFP fluorescence can be detected by RT-PCR and in both melon roots and leaves by confocal scanning laser microscopy. Here, We constructed MNSV infections clone and pMNSV-cp-GFP clone vectors for the first time in China. The MNSV infections clone provided a tool for the analysis of its pathogenic mechanism at the molecular level. In addition, we also constructed MNSV infectious clone vectors carrying GFP, which could be used in the fluorescent labeling of MNSV and its distribution, movement and colonization during virus infection.     

Functional degeneration of the resistance gene nsv against Melon necrotic spot virus at low temperature

The single recessive gene, nsv, which confers resistance against Melon necrotic spot virus (MNSV), has recently been used to develop virus-resistant melon cultivars in Japan. However, the Chiba isolate of MNSV, a common isolate in Japan, infected resistant cultivars when inoculated melon plants were grown at 15°C. Viral RNAs accumulated in protoplasts from resistant cultivars at both 15 and 20°C. Mechanical inoculation of the cotyledons caused MNSV to spread throughout the leaves at 15°C, but not at 20°C. These results support our novel hypothesis that a temperature-sensitive inactivation of disease resistance genes occurs at the nsv locus in melon cultivars with the resistance gene grown at temperatures below 20°C. The first and second authors contributed equally to this research.     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

Frequency of Alternaria brassicicola in commercial cabbage seeds in Japan

Plug seedlings, widely used in cabbage cultivation in Japan, are often infected by seed-borne pathogens, especially the serious pathogen Alternaria brassicicola. Because information on seed infestation is scant in Japan, we investigated fungal infestation in commercial batches of cabbage seeds produced between 1984 and 2001. A total of 123 lots were divided into six groups by production period (1984–1989, 1994–1998, and 2001) and by use or nonuse of fungicide. One hundred seeds from each lot were incubated separately on agar at 25°C to isolate the predominant fungus. Alternaria brassicicola was isolated most frequently, 0%–94% of the seeds depending on seed lot or 6%–21% of the seeds grouped by production period and fungicide treatment. Thus, the pathogen was isolated even from seeds refrigerated for 17 years. Alternaria brassicicola accounted for 57%–95% of all isolated fungi by the group and was higher on older or fungicide-treated seeds. Seeds that were not treated with fungicide in lots grouped by production districts in western Japan were infested with A. brassicicola at a rate of over 12%, higher than that in the eastern region (<4%). Infestation was higher in the warmer areas of Japan. Eighty-five isolates, other than A. brassicicola, produced spots on cabbage cotyledons, although they were not isolated as frequently: less than 5% of seeds by group separated by production period and fungicide treatment. Most of these isolates were Alternaria alternata. This is the first report on the frequency of fungal infestation of commercial cabbage seeds in Japan.     

Isolation of Microdochium oryzae and Pinatubo oryzae from Rice Seeds and their Survival on Stored Seeds

The aim of these studies was to develop a semi-selective medium to differentiate Microdochium oryzae and Pinatubo oryzae, determine the frequency of seed infection of M. oryzae, study survival of the pathogen in stored seeds, and determine the frequency of infection of seed components. To simulate epidemics of differing intensities, panicles of rice cultivars that are susceptible (IR36) and resistant (IR42 and IR46) to M. oryzae were either non-inoculated, inoculated once, twice, or three times with a conidial suspension of M. oryzae. Both M. oryzae and P. oryzae colonies were recovered from seeds and were similar in culture. A semi-selective medium developed to detect M. oryzae seed infection rates aided in differentiating M. oryzae and P. oryzae by stimulating aerial conidiogenesis of P. oryzae. The conclusions taken from these results were: (a) seeds of IR36 had higher infection of M. oryzae than of IR42 and IR46 from plants grown in the dry season, but had lower infection of M. oryzae than of IR42 and IR46 from plants grown in the wet season; (b) M. oryzae infected seeds increased with an increase in the epidemic intensity with the highest occurring after three inoculations, the least occurring with non-inoculation, and intermediate with one, or two inoculations; (c) survival of M. oryzae decreased over time in seed lots stored at 10°C and 40% relative humidity and (d) all components of the rice seeds of IR36, IR42 and IR46 lots were infected with M. oryzae with the highest frequency in the endosperm and lemma, intermediate in the basal glumes and palea, and the least in the embryo.     

两种甜瓜病毒寿光分离物的分子检测与鉴定

甜瓜坏死斑点病毒(Melon necrotic spot virus,MNSV)及瓜类褪绿黄化病毒(Cucurbit chlorotic yellows virus,CCYV)近年来在瓜类种植区均大面积发生,危害较为严重,已成为制约甜瓜生产的重要因素。本研究广泛收集疑似感染MNSV及CCYV的甜瓜病叶,从中提取植物总RNA进行RT-PCR扩增,将产物分别连接到pEASYT1Simple克隆载体上,对含有目的片段的重组子进行测序及比对分析。结果显示得到了与预期结果一致的DNA序列,其扩增产物大小分别为673bp(MNSV)和877bp(CCYV)。同源性分析结果表明,MNSV和CCYV寿光分离物的核苷酸序列与中国其他地区或一些国家已报道的分离物同源性达99%~100%。     

Solar UV-B radiation limits seedborne anthracnose infection and induces physiological and biochemical responses in Lupinus mutabilis

The aim of this study was to test whether solar UV-B radiation and temperature have an effect on infection of lupin seeds by Colletotrichum acutatum. Samples of infected seed were placed in a solar oven and exposed on sunny days for 15, 30, 45, 60, 75, 90 and 120 min. The degree of reduction in disease incidence and seed germination was dependent on the exposure time. Exposure times of 75 min (UV-B 4.41 kJ m⁻², ≈76 °C) and higher reduced incidence from 5% to undetectable levels, but also reduced seed germination by around 10% compared with untreated seed. Therefore, in a second experiment, infected seeds were exposed for 45 or 60 min in the solar oven (UV-B 2.83 or 3.75 kJ m⁻², respectively, ≈76 °C), for 60 min at ambient temperature (UV-B 3.75 kJ m⁻², ≈21 °C) or to dry heat for 60 min at 75 °C. Exposure for 60 min in the solar oven reduced seed infection by 99%, while UV-B-radiated seed at ambient temperature or dry-heat reduced infection by 60% or 32%, respectively. To evaluate the effect of UV-B plus high temperature on seedlings, lupin seed exposed for 45 or 60 min (UV-B 2.83 or 3.75 kJ m⁻², respectively, at ≈76 °C) were grown and physiological and biochemical responses of the seedlings were assessed. Seedlings from exposed seed had higher total concentrations of chlorophyll, protein and peroxidase activity than those grown from unexposed infected seed.     

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.     

Occurrence of melon necrotic spot carmovirus in Italy

Since 1995, a severe necrosis disease has been observed in winter melon ( Cucumis melo var. inodorus ) grown in the open in Sardegna region. At the stage of fruit ripening, infected plants show decline and premature death, a syndrome known as'collapse'. Fungal pathogens have never been detected. In 1998, melon necrotic spot carmovirus (MNSV) was isolated from diseased plants showing necrotic symptoms on leaves and branches. The virus was identified by biological and serological assays. MNSV may represent a limiting factor to winter melon production in Sardegna where the'inodorus'group is widely cultivated and appreciated for local consumption. This is the first occurrence of MNSV in Italy.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

Seed disinfection treatments do not sufficiently eliminate the infectivity of Cucumber green mottle mosaic virus (CGMMV) on cucurbit seeds

Tobamoviruses induce crop diseases that are responsible for significant economic losses around the world. Like other tobamoviruses, Cucumber green mottle mosaic virus (CGMMV) forms highly stable particles that can persist for long periods on plant debris, in soil and on seed surfaces. These particles serve as a primary source of infection, infecting seedlings from which the virus can then be mechanically transmitted to other neighbouring plants. Contaminated seeds also provide a route for the movement of the virus between countries and its introduction into new areas. Effective seed disinfection treatments and the use of uncontaminated seed may reduce the global prevalence of this virus. Several treatments based on the use of heat or chemicals have been reported to effectively eliminate CGMMV and other tobamoviruses from seeds. An evaluation of these treatments on highly contaminated seed lots revealed inconsistent results, which encouraged the construction of a more accurate detection method that combines morphological, serological, molecular and biological analyses in one protocol. The detection of viable (infectious) viral particles in seed treated with heat, trisodium phosphate or a combined treatment, indicates that these treatments are insufficient. The serological detection of CGMMV in the inner parts of infected seeds provides a possible explanation for the inconsistent efficacy of these treatments.     

The role of seed-borne Aspergillus niger in transmission of black mould of onion

The incidence of Aspergillus niger (black mould) was found to be high on seed produced in hot (desert) climates. The fungus was transmitted from contaminated seeds to the cotyledons and first true leaves of seedling onions grown under controlled conditions. Seedlings grown from contaminated seeds had longer roots but shorter shoots than those grown from healthy seeds. Seed treatment with either benomyl plusthiramat the rate of 2.5 + 2.5 g active ingredient/kg seed or in hot water (15 min immersion at 60°C) eliminated the fungus in a naturally infected seed stock, untreated seed of which gave 30% infected seedlings.     

Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae , is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30-cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2-m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR-specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.     

Detection of Acidovorax valerianellae in corn-salad seeds, seed transmission of the pathogen and disease development in the field

Acidovorax valerianellae , the causal agent of bacterial black spot of corn salad and responsible for severe economic losses to this vegetable in France, was successfully transmitted to corn-salad plants by artificially inoculated seeds in glasshouse and field experiments. In the field experiments, climatic data recorded under plastic tunnels indicated that increasing temperature and relative humidity increased symptom development. To investigate the possible contamination of commercial seedlots of corn salad, a seed test was developed consisting of soaking batches of seeds (five batches each of 5000, 1000, 500 and 100 seeds) overnight at 4°C in distilled sterile water, followed by dilution-plating of seed extracts on TSAV (tryptic soya agar for A. valerianellae ) semiselective medium. Suspected colonies were identified by biochemical and pathogenicity tests or, within 24 h, using antibodies specific to A. valerianellae . Acidovorax valerianellae was detected in three lots. Seed infection levels ranged from 0·10 to 0·89% of contaminated seeds and a single seed carried up to 1800 A. valerianellae colony-forming units.