Detection and Location of Seed-Borne Inoculum of Didymella bryoniae and its Transmission in Seedlings of Cucumber and Pumpkin

Of the 91 tested cucurbit seed samples from thirteen countries nine from four countries were found to be infected with Didymella bryoniae. The pathogen is located on and in the seed coat including the perisperm and in the tissue of the cotyledons. Primary seedling infection occurred on the radicle, hypoctyl and cotyledons. Infection of the radicle generally caused pre-emergence tot while infection on the hypocotyl and cotyledons developed furthere inoculum for infection of the first true leaves and the stem. Experimentally, all the isolates of D. bryoniae could infect cucumber (Cucumis sativus), oriental melon (Cucumis melo var. makuwa), pumpkin (Cucurbita pepo) and watermelon (Citrullus vulgaris) at different growth stages; the susceptibility of cucumber and pumpkin was markedly influenced by previailing humid conditions.The blotter method was found more suitable for detection of seed-borne infection than the agar plate method.     

Alternaria on cruciferous plants. 4.Alternaria species on seed of some cruciferous crops and their pathogenicity

The incidence ofAlternaria spp. on seed samples of cruciferous vegetable crops was surveyed between 1990 and 1992. Some commercial seed lots of crucifers which are commonly grown in Japan were infested withAlternaria species. ThreeAlternaria species were encountered on the seed samples ofBrassica campestris, B. orelacea, andRaphanus sativus. The most frequently detected species wereA. japonica andA. alternata onB. campestris, A. brassicicola onB. oleracea, andA. japonica andA. alternata onR. sativus, respectively.Alternaria brassicae was not detected in this study.Alternaria brassicicola isolates from these crops produced necrotic lesions on all of the crucifer seedlings inoculated, whileA. japonica induced different reactions in different plants or plant parts depending on isolates used in inoculation tests. In contrast, most isolates ofA. alternata could not produce necrotic lesions on foliage leaves of crucifers inoculated, although some of them produced clear lesions only on cotyledons.Alternaria alternata associated with these cruciferous crop seeds was considered to be an oppotunistic parasite of these crops.     

Development and characterization of microsatellite markers in Cucumis

This study provides a set of useful SSR markers and describes their development, characterization and application for diversity studies.Sixty one Cucumis SSR markers were developed, most of them (46) from melon (Cucumis melo L.) genomic libraries. Forty of the markers (30 melon and 10 cucumber SSRs) were evaluated for length polymorphism in a sample of 13 melon genotypes and 11 cucumber (Cucumis sativus L.) genotypes. PCR-amplification revealed up to six size alleles among the melon genotypes and up to five alleles among the cucumber genotypes, with mean gene-diversity values of 0.52 and 0.28 for melon and cucumber, respectively. These differences are in accordance with the known narrower genetic background of the cucumber. SSR data were applied to phylogenetic analysis among the melon and cucumber genotypes. A clear distinction between the ’exotic’ groups and the sweet cultivated groups was demonstrated in melon. In cucumber, separation between the two sub-species, C.sativus var. sativus and C.sativus var. hardwickii,was obtained. Conservation of SSR loci between melon and cucumber was proven by sequence comparisons. Received: 17 April 2000 / Accepted: 16 May 2000     

Plant-mediated interactions between insects and a fungal plant pathogen and the role of plant chemical responses to infection

Diverse organisms simultaneously exploit plants in nature, but most studies do not examine multiple types of exploiters like phytophagous insects and fungal, bacterial, and viral plant pathogens. This study examined patterns of induction of antipathogenic peroxidase enzymes and phenolics after infection by the cucurbit scab fungus, Cladosporium cucumerinum, and then determined if induction mediated ecological effects on Colletotrichum orbiculare, another fungal pathogen, and two insect herbivores, spotted cucumber beetles, and melon aphids. Peroxidase induction occurred in inoculated, `local,' symptom-bearing leaves 3 days after inoculation, and in `systemic,' symptom-free leaves on the same plants 1 day later. Phenolics were elevated in systemic but not in local leaves 3 days after inoculation. Detached systemic leaves from plants inoculated with C. cucumerinum developed significantly fewer and smaller lesions after challenge with C. orbiculare. Spotted cucumber beetles did not show consistently significant preferences for infected versus control leaf disks in comparisons using local or systemic leaves, but trends differed significantly between leaf positions. In no-choice tests, beetles removed more leaf area from local but not from systemic infected leaves compared to control leaves, and melon aphid reproduction was enhanced on local infected leaves. In the field, cucumber beetle and melon aphid densities did not differ between infected and control plants. Antipathogenic plant chemical responses did not predict reduced herbivory by insects. Other changes in metabolism may explain the positive direction and spatially dependent nature of plant-mediated interactions between pathogens and insects in this system. Received: 28 September 1997 / Accepted: 9 February 1998     

An outbreak of yellow mold of peanut seedlings in Texas

Yellow mold of peanut (Arachis hypogaea) seedlings caused by Aspergillus flavus was first observed during May 1984 in a commercial peanut farm in south Texas. The mold caused preemergence rotting of peanut seed and seedlings. On emerged seedlings the infection was largely restricted to cotyledons. The diseased plants were chlorotic, stunted, and leaflets were reduced in size with pointed tips and vein-clearing. Aflatoxins were found in cotyledons of infected seedlings but not in roots, hypocotyls, or leaves. A. flavus was the predominant fungus in the seed lot planted by the grower. Six isolates of A. flavus isolated from the seed and diseased seedlings were pathogenic to peanut in greenhouse tests.Texas Agriculture Experiment Station No. TA 20319 and ICRISAT Journal Article No. JA 614.     

Damping-off of Some Cucurbitaceous Crops in Saudi Arabia with Reference to Control Methods

Isolation of seed borne fungi from sweet melon (najed and red Queen varieties) and vegetable marrow (squash) using PDA, Plain agar media and blotters revealed the presence of Alternaria alternata (Fr.) Keissler, Fusarium semitectum var,majus from Najed melon, A. alternata (Fr.) Keissler, F. sambucinum Fuckel from Red Queen melon and A. chlamydospora Mouchacca, Cephalosporium sp., and F. oxysporum schlecht from squash. Pathogenicity tests showed, that all these fungi were highly pathogenic on their respective hosts. The optimum temperature for its growth ranged from 25–30°C and the optimum pH was 6.0. In pot trials, seed dressing with Banrot, Bavistin and Topsin-M at the rate of 4 g/kg seed was superior in controlling the damping-off of melon and squash. These fungicides were effective in inhibiting mycelial growth, spore germination and development of the isolated fungi. Hot water treatment at 55 C/20 min or solar heating (av. 37°C) for 90 min were next to fungicides.     

Inheritance of organelle DNA markers in a pea cross associated with nuclear-cytoplasmic incompatibility

An unusual biparental mode of plastid inheritance was found in pea, in a cross associated with nuclear-cytoplasmic incompatibility manifested as deficiency of chlorophyll pigmentation. Plastid DNA marker trnK and mitochondrial DNA marker cox1 were analyzed in F1 progeny that received cytoplasm from an accession of a wild subspecies Pisum sativum ssp. elatius. Plants with sectors of green tissue on leaves and seed cotyledons with green patches on an otherwise chlorotic background were found to carry paternally inherited plastid DNA, suggesting that photosynthetic function was affected by nuclear-cytoplasmic conflict and required proliferation of paternally inherited plastids for normal performance. The paternally inherited plastid DNA marker was also observed in the roots. The presence of the paternal marker in cotyledons, roots and leaves was independent of each other. Inheritance of the mitochondrial DNA marker cox1 appeared to be of the maternal type.     

Apple mosaic virus isolated from hop plants in Japan

A strain of apple mosaic virus was isolated from hop plants in Japan. The virus was purified from young hop plants and back-inoculated to virus-free hop plants obtained by meristem tip culture. Inoculated plants developed chlorotic spots, ringspots and a band pattern accompanied by necrosis in the inoculated and systemically infected leaves. Shoot tips of infected plants sometimes became necrotic and these symptoms resembled those of a ring- and band-pattern mosaiclike disease prevalent in hop gardens in Japan. Since apple mosaic virus was recovered from infected plants, it is likely that the virus was the causal agent of this disease. Agar gel double diffusion tests and ELISA showed the hop virus to be serologically closely related to apple mosaic virus (ApMV), and distantly related to prunus necrotic ringspot virus (PNRSV). The virus had a narrow host range, and infected only cucumber of 18 species of Cucurbitaceae, Chenopodiaceae, Leguminosae or Solanaceae inoculated. It produced chlorotic spots on the inoculated cotyledons of cucumber, but no systemic infection. By contrast, ApMV from apple and PNRSV from peach had wide host ranges and infected cucumber plants systemically.     

First report of Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler on Sonchus oleraceus L. and Convolvulus arvensis L. in Algeria

Alternaria leaf spot caused by Alternaria alternata (Fries.) Kiessler was found on sow thistle (Sonchus oleraceus L.) and field bindweed (Convolvulus arvensis L.) in the experimental station of ENSA (Ecole Nationale Supérieure d'Agronomie) in Algiers, Algeria, during the winter of 2016. Necrotic spots in the form of concentric circles were observed on the leaves of both weeds with disease incidence of approximately 70% and disease severity ranging from 50% to 70%. Fungi were isolated from the infected leaves and identified as A. alternata, based on morphological and molecular analyses (using genetic marker internal transcribed spacer, ITS of rDNA). Pathogenicity tests confirmed that A. alternata is the causing agent of leaf spot disease of sow thistle and field bindweed in accordance with the original symptoms. To the best of our knowledge, this is the first report of sow thistle and field bindweed naturally infected by A. alternata in Algeria.     

Water stress in cucumber cotyledons infected withXanthomonas campestris pv.cucurbitae

Xanthomonas compestris pv.cucurbitae strain 62 was isolated from diseased cucumber seedlings grown in an open field in Egypt and identified in this study. Inoculation of cucumber seedlings with this strain resulted in reduction of relative water content (RWC) and increase in free proline content of infected cotyledons. RWC was found to be decreased by the increase of disease severity. The reverse was true for free proline content which was found to be increased by the increase of disease severity. These results indicate that water stress increased in direct proportion to the disease severity. Highly significant increase in free proline content was found to occur in organs of cucumber seedlings inoculated with strain 62 and showing disease symptoms on cotyledons only. This implied that water stress was not limited to symptomatic cotyledons but extended to the whole seedling.     

Vertical transmission of fungal endophytes is widespread in forbs

To date, it has been thought that endophytic fungi in forbs infect the leaves of their hosts most commonly by air‐borne spores (termed “horizontal transmission”). Here, we show that vertical transmission from mother plant to offspring, via seeds, occurs in six forb species (Centaurea cyanus, C. nigra, Papaver rhoeas, Plantago lanceolata, Rumex acetosa, and Senecio vulgaris), suggesting that this may be a widespread phenomenon. Mature seeds were collected from field‐grown plants and endophytes isolated from these, and from subsequent cotyledons and true leaves of seedlings, grown in sterile conditions. Most seeds contain one species of fungus, although the identity of the endophyte differs between plant species. Strong evidence for vertical transmission was found for two endophyte species, Alternaria alternata and Cladosporium sphaerospermum. These fungi were recovered from within seeds, cotyledons, and true leaves, although the plant species they were associated with differed. Vertical transmission appears to be an imperfect process, and germination seems to present a bottleneck for fungal growth. We also found that A. alternata and C. sphaerospermum occur on, and within pollen grains, showing that endophyte transmission can be both within and between plant generations. Fungal growth with the pollen tube is likely to be the way in which endophytes enter the developing seed. The fact that true vertical transmission seems common suggests a more mutualistic association between these fungi and their hosts than has previously been thought, and possession of endophytes by seedling plants could have far‐reaching ecological consequences. Seedlings may have different growth rates and be better protected against herbivores and pathogens, dependent on the fungi that were present in the mother plant. This would represent a novel case of trans‐generational resistance in plants.     

Inoculation of cucumber,melon and zucchini varieties with Tomato leaf curl New Delhi virus and evaluation of infection using different detection methods

The disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post‐inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.     

Comparative mapping of ZYMV resistances in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.)

Zucchini yellow mosaic virus (ZYMV) routinely causes significant losses in cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). ZYMV resistances from the cucumber population TMG1 and the melon plant introduction (PI) 414723 show different modes of inheritance and their genetic relationships are unknown. We used molecular markers tightly linked to ZYMV resistances from cucumber and melon for comparative mapping. A 5-kb genomic region (YCZ-5) cosegregating with the zym locus of cucumber was cloned and sequenced to reveal single nucleotide polymorphisms and indels distinguishing alleles from ZYMV-resistant (TMG1) and susceptible (Straight 8) cucumbers. A low-copy region of the YCZ-5 clone was hybridized to bacterial artificial chromosome (BAC) clones of melon and a 180-kb contig assembled. One end of this melon contig was mapped in cucumber and cosegregated with ZYMV resistance, demonstrating that physically linked regions in melon show genetic linkage in cucumber. However the YCZ-5 region segregated independently of ZYMV resistance loci in two melon families. These results establish that these sources of ZYMV resistances from cucumber TMG1 and melon PI414723 are likely non-syntenic.     

The Infection of Festuca arundinacea by Puccinia graminis subsp. graminicola

Puccinia graminis subsp. graminicola has caused economically important losses of tall fescue (Festuca arundinacea Schreb.) in North America. This rust infects leaves, culms, and spikelets of the host. Initital symptoms consist of very small, chlorotic flecks, followed by long, narrow lesions. Invasive hyphae were diffuse and filamentous, changing to dense and blocky with the formation of hymenia. Tissues were extensively colonized, including development caryopses. Seedborne infection consisted of mycelium within the embryo and urediniospores carried on both surfaces of the glumes. It is not known whether caryopses that are internally infected can germinate to produce infected individuals, but this could be an important quarantine consideration. Although, at the ultrastructural level, urediniospore development in this fungus is similar to that of other rusts, we detected an additional layer at the interface of the urediniospore and its pedicel. This layer may play a role in the, release of urediniospores from their pedicels.     

Effect of Wind-induced Mechanical Stress on Soluble Peroxidase Activity and Resistance to Pests in Cucumber

Wind-induced mechanical stress (MS) significantly increased the soluble peroxidase activity in leaves of cucumber over control levels after 9 days of treatment. In comparison, inoculation with the fungal pathogen Cladosporium cucumerinum induced significant increases in peroxidase after 4 days. Cucurbit anthracnose symptom development caused by Colletotrichum orbiculare was greater on leaves of seedlings exposed to 6 but not to 9 or 12 days of wind-induced MS than on leaves of control plants. In contrast, reproduction of melon aphids was significantly reduced on leaves exposed to 12 days of MS relative to controls. These results indicate that wind-induced MS can induce soluble peroxidase activity in cucumber and have divergent effects on the resistance to insects and pathogens.     

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a fulllength cDNA predicts a polypeptide of 74397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.     

Different enhancement of senescence induced by metabolic products of Alternaria alternata in tobacco leaves of different ages

The purpose of this study was to explore the mechanisms by which Alternaria alternata damages tobacco (Nicotiana tabacum) leaves. Treatment with A. alternata metabolic products enhanced senescence in leaves of different ages, as indicated by the significant decrease in chlorophyll, soluble protein, photosynthetic O₂ evolution and catalase (CAT, EC 1.11.1.6) activity as well as an increase in H₂O₂ content. The induction of senescence by A. alternata metabolic products increased as the age of the leaves increased. A. alternata metabolic products greatly influenced the behavior of photosystem II (PSII) in the leaves: oxygen evolving complex (OEC) activity and electron transport from primary quinone electron acceptor of PS II (Q_A) to secondary quinone electron acceptor of PS II (Q_B) were both significantly inhibited. This inhibition also became more pronounced in older leaves. In vitro experiments revealed that, without the influence of natural senescence, the A. alternata metabolic products directly inhibited the activity of a commercial CAT solution and inhibited photosynthetic O₂ evolution, which resulted in excess PSII excitation pressure and an overaccumulation of H₂O₂ in leaf segments. These results suggest that the significant declines in photosynthesis and CAT activity induced by the metabolic products of A. alternata were important contributors to the overaccumulation of reactive oxygen species (ROS), which accelerated senescence in tobacco leaves. The fact that the enhancement of senescence was getting more pronounced with the age of tobacco leaves might be related to the fact that older leaves already had higher H₂O₂ levels and less antioxidant activity as reflected in lower CAT activity.     

Sensibilité du Melon àSphaerotheca fuliginea en Fonction de L'Etage Foliaire

Effect of leaf position on the susceptibility of melon plants to artificial infection with powdery mildew, Sphaerotheca fuliginea The leaf position of melon plants seems to play a role on their susceptibility when they are artificially infected with powdery mildew Sphaerotheca fuliginea. The cotyledons are generally very susceptible, while the first leaf relatively resistant; the susceptibility again continues up to the 4th–5th leaf (but less susceptible than the cotyledons) and then after it decreases; these results can be obtained on plants in greenhouse or on detached leaves in Petri dishes. From this observation, we think that the screening of melon genotypes for resistance to powdery mildew can be evaluated neither on the cotyledons which are very susceptible nor on the first leaf which is resistant, but on the third leaf which is moderately susceptible. In fact, there is a good correlation between the reaction of the third leaf and the resistance or susceptibility of genotypes.     

A Cucumber Disease Caused by Alternaria alternata and its Control

A severe leaf spot disease of cucumber caused by a pathotype of Alternaria alternata (Fr.) Keissler was recorded recently in plastic houses in Crete. Lesions ranged in size of a pin point to over 5 cm in diameter, with necrotic tissue on most of their area and a surrounding yellow zone. The pathogen grew satisfactorily on PDA at temperatures between 5 °C–40 °C and spore germination occurred in the range less than 10 °C to over 37 °C. Optimum temperature in both cases was near 26 °C. Of,13 fungicides tested in vitro, sodium omadine, etem, dichlofluanid, captan and folpet were the most inhibitory on spore germination, and iprodione, sodium omadine and dichlofluanid on mycelial growth. Of 25 fungicides applied on two leaf cucumber plants 24 h before inoculation, maneb, etem, dichlofluanid and chlorothalonil were the most effective. When the last fungicides, plus mancozeb, were applied 24 h after inoculation only maneb was effective. In greenhouse experiments, iprodione, prochloraz-manganese-complex, chlorothalonil, dichlofluanid, guazatine, maneb and etem were the most effective for disease control, while mancozeb was less effective. The local cucumber cv. Knossos and the Dutch F₁ hybrids Evadan, Frella, Herta, Malfa, Mazourka, Pepinex 69 and Renova were all susceptible to infection.