Resistance in melon to Monosporascus cannonballus and M. eutypoides: Fungal pathogens associated with Monosporascus root rot and vine decline

The fungal species Monosporascus cannonballus and M. eutypoides have been described as the causal agents of Monosporascus root rot and vine decline disease (MRRVD), which mainly affects melon and watermelon crops. Resistance to M. cannonballus has been reported in some melon cultivars (ssp. melo). Moreover, melon ssp. agrestis accessions have proven to be better resistance sources. This is the case of the Korean accession ‘Pat 81’, highly resistant under field and artificial inoculation. The objective of the work here presented was the evaluation of the resistance to MRRVD of different accessions representing the variability of Cucumis melo ssp. agrestis, against both, M. cannonballus and M. eutypoides, in a multiyear assay under different infection conditions. In general, M. eutypoides was less aggressive than M. cannonballus in the different environmental conditions. There was a strong influence of temperature on MRRVD, with more severe symptoms with higher temperatures and with variable effect of infection on plant development depending on the fungal species considered. Resistance to MRRVD has been confirmed in ‘Pat 81’ and in its derived F1 with a susceptible Piel de Sapo melon. Among the new germplasm explored, African accessions (both wild agrestis and exotic cultivated acidulus) showed good performance in artificial inoculation assays and in field conditions. These sources do not present compatibility problems with commercial melons, so they can be introduced in backcrossing programs. The accession assayed of the wild relative Cucumis metuliferus, also resistant to Fusarium wilt and to root-knot nematode, was highly resistant to MRRVD. The interest of this accession mainly relies in its advantages as a rootstock for melon.     

Melon Fruits: Genetic Diversity,Physiology, and Biotechnology Features

Among Cucurbitaceae, Cucumis melo is one of the most important cultivated cucurbits. They are grown primarily for their fruit, which generally have a sweet aromatic flavor, with great diversity and size (50 g to 15 kg), flesh color (orange, green, white, and pink), rind color (green, yellow, white, orange, red, and gray), form (round, flat, and elongated), and dimension (4 to 200 cm). C. melo can be broken down into seven distinct types based on the previously discussed variations in the species. The melon fruits can be either climacteric or nonclimacteric, and as such, fruit can adhere to the stem or have an abscission layer where they will fall from the plant naturally at maturity. Traditional plant breeding of melons has been done for 100 years wherein plants were primarily developed as open-pollinated cultivars. More recently, in the past 30 years, melon improvement has been done by more traditional hybridization techniques. An improvement in germplasm is relatively slow and is limited by a restricted gene pool. Strong sexual incompatibility at the interspecific and intergeneric levels has restricted rapid development of new cultivars with high levels of disease resistance, insect resistance, flavor, and sweetness. In order to increase the rate and diversity of new traits in melon it would be advantageous to introduce new genes needed to enhance both melon productivity and melon fruit quality. This requires plant tissue and plant transformation techniques to introduce new or foreign genes into C. melo germplasm. In order to achieve a successful commercial application from biotechnology, a competent plant regeneration system of in vitro cultures for melon is required. More than 40 in vitro melon regeneration programs have been reported; however, regeneration of the various melon types has been highly variable and in some cases impossible. The reasons for this are still unknown, but this plays a heavy negative role on trying to use plant transformation technology to improve melon germplasm. In vitro manipulation of melon is difficult; genotypic responses to the culture method (i.e., organogenesis, somatic embryogenesis, etc.) as well as conditions for environmental and hormonal requirements for plant growth and regeneration continue to be poorly understood for developing simple in vitro procedures to culture and transform all C. melo genotypes. In many cases, this has to be done on an individual line basis. The present paper describes the various research findings related to successful approaches to plant regeneration and transgenic transformation of C. melo. It also describes potential improvement of melon to improve fruit quality characteristics and postharvest handling. Despite more than 140 transgenic melon field trials in the United States in 1996, there are still no commercial transgenic melon cultivars on the market. This may be a combination of technical or performance factors, intellectual property rights concerns, and, most likely, a lack of public acceptance. Regardless, the future for improvement of melon germplasm is bright when considering the knowledge base for both techniques and gene pools potentially useable for melon improvement.     

Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon

In melon, the Fom-1 gene confers monogenic resistance against the soil-borne fungus Fusarium oxysporum f. sp. melonis, races 0 and 2, while the closely linked Prv gene specifies resistance against the papaya ring spot virus. Markers linked to these resistance (R) genes were identified using two recombinant inbred line populations, derived from crosses between Cucumis melo Védrantais and C. melo PI 161375, and between C. melo Védrantais and C. melo PI 414723, respectively. Using bulked segregant analysis, as well as systematic scoring of the mapping populations, we developed two amplified fragment length polymorphism markers, two random amplified polymorphic DNA markers and five restriction fragment length polymorphism (RFLP) markers linked to this locus. Four of the RFLP sequences bear homology to nucleotide-binding site–leucine-rich repeat R genes, indicating the presence of a significant R-gene cluster in this locus. Our study provides the most closely linked markers published so far for these important traits. It also improves the resolution of the whole linkage group IX, which was difficult to order in our previous studies. Two of the markers were converted to cleaved amplified polymorphic sequence markers to facilitate their application in marker-assisted selection. Testing these two markers in several melon lines revealed different marker haplotypes in the melon germplasm and supported multiple, independent origin of the Fusarium races 0 and 2 resistance trait.     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.     

Association of tomato leaf curl Palampur virus with yellow mosaic disease of Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) in India

Natural occurrence of yellow mosaic disease was observed on Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) with disease incidences of ~36 and ~27%, respectively. Association of tomato leaf curl Palampur virus (ToLCPV) with the disease was investigated by Polymersae chain reaction (PCR) using begomovirus-specific primers. Full-length genome was amplified by rolling circle amplification (RCA) method from representative samples of C. melo and C. callosus. RCA products obtained were cloned and sequenced. Analyses of sequence data revealed the presence of full-length begomoviral genome of 2756 nucleotides with the gene arrangement of a typical begomovirus: HQ848383 (C. melo) and GU253914 (C. callosus). Both the isolates shared 99% sequence identity together and high 97–99% identities and the closest phylogenetic relationships with ToLCPV strains reported worldwide, hence identified as two new members of ToLCPV. Natural occurrence of ToLCPV on C. melo and C. callosus is the first report.     

Evaluation of Streptomyces spp. for biocontrol of gummy stem blight (Didymella bryoniae) and growth promotion of Cucumis melo L.

Gummy stem blight of Cucumis melo L. (melon) caused by Didymella bryoniae is a serious disease in the major production area of northwest China. Two Streptomyces isolates (Streptomyces pactum A12 and S. globisporus subsp. globisporus C28) previously isolated from the Qinghai-Tibet Plateau were investigated regarding their biocontrol of gummy stem blight and growth promotion of melon under controlled conditions. Streptomyces A12 and C28 indicated obvious antagonistic activity against D. bryoniae in vitro. Both A12 and C28 significantly decreased disease severity and AUDPC (area under the disease progress curve) of melon gummy stem blight in vivo (P < 0.05). Ten-fold dilution of C28 culture filtrate was more effective in controlling the disease compared with other treatments, the disease reduction effects were 41.0–64.2%. The mean fresh weights were increased by 40.4% for plants, 44.2% for roots, and 40.3% for aerial parts, when A12 was applied in both nursery soil and transplanted soil. Streptomyces C28 also increased the mean fresh weights of melon plants by 18.4–49.0% compared with the control in pot trial. Streptomyces A12 and C28 showed substantial colonization abilities in the rhizosphere and on the rhizoplane of melon plants. Results demonstrated that Streptomyces A12 and C28 were of positive effect on the biocontrol of gummy stem blight and growth promotion of Cucumis melo L.     

Transformation of a marker-free and vector-free antisense ACC oxidase gene cassette into melon via the pollen-tube pathway

Purpose of work  

Melons have short shelf-lives due to fruit ripening caused by ethylene production. The 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene is essential for ethylene biosynthesis. As fruit ripening in other fruit crops can be deterred by down-regulation of ACC oxidase expression, we have carried out similar work to improve fruit quality and shelf-life of the melon Cucumis melo.     

Zucchini Yellow Fleck Virus is an Emergent Virus on Melon in Sicily (Italy)

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin‐point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme‐linked immunosorbent assay to the most important viruses‐infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV‐infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.     

Plant Growth‐Promoting Bacteria from Solarized Soil with the Ability to Protect Melon Against Root Rot and Vine Decline Caused by Monosporascus cannonballus

This study was undertaken to isolate indigenous plant growth‐promoting (PGP) bacteria from solarized soil effective in the biocontrol of Monosporascus cannonballus, the cause of root rot and vine decline of melon, which is one of the most destructive soilborne diseases of this crop worldwide. The screening strategy resulted in the selection of two interesting PGP bacteria as biocontrol candidates against M. cannonballus belonging to the same microbial community. The two bacterial species, identified according to phenotypic, physiological tests and analysis of the 16S rDNA sequence as Bacillus subtilis/amyloliquefaciens (BsCR) and Pseudomonas putida (PpF4), showed PGP traits and in vitro antagonistic activity towards M. cannonballus. Antagonism by BsCR was characterized by a consistent inhibition of the pathogen in vitro growth; PpF4 strongly inhibited the development of perithecia of the pathogen. Under greenhouse conditions, the selected bacteria were tested for their biocontrol activity in the pathosystem melon‐M. cannonballus. BsCR alone and in combination with PpF4 determined a consistent decrease in the disease symptoms. BsCR and the combination of the bacterial strains significantly increased root biomass in both inoculated and un‐inoculated plant. Upon seed treatment with BsCR, the accumulation and isoenzyme induction of peroxidase in roots as biochemical marker for induction of resistance were found, thus indicating that BsCR may reduce the disease severity also by the activation of the plant defence responses. The study highlights the synergistic biocontrol potential of B. subtilis BsCR and P. putida PpF4 in the integrated management of root rot and vine decline of melon caused by M. cannonballus.     

Effect of modified endogenous ethylene production on sex expression, bisexual flower development and fruit production in melon (Cucumis melo L.)

Members of the Cucurbitaceae family display a range of sexual phenotypes including various combinations of male, female, or bisexual flowers. Ethylene appears to be a key hormone regulating the sex determination process. Application of ethylene, or inhibition of ethylene action, increases or decreases the number of pistil-bearing buds, respectively. Elevated levels of ethylene production and expression of genes for ethylene biosynthesis, have been correlated with pistillate flower production. In this study, we sought to determine the effect of modified endogenous ethylene production on sex expression by constitutively expressing ACS (1-aminocyclopropane-1-carboxylate synthase), the first committed enzyme for ethylene biosynthesis, in transgenic melons (Cucumis melo L.). Most melon genotypes are andromonoecious, where an initial phase of male flowers is followed by a mixture of bisexual and male flowers. ACS melon plants showed increased ethylene production by leaves and flower buds, and increased femaleness as measured by earlier and increased number of bisexual buds. ACS melons also had earlier and increased number of bisexual buds that matured to anthesis, suggesting that ethylene is important not only for sex determination, but also for development of the bisexual bud to maturity. Field studies showed that ACS melons had earlier mature bisexual flowers, earlier fruit set, and increased number of fruit set on closely spaced nodes on the main stem. These results provide a direct demonstration of the importance of endogenous ethylene production for female reproductive processes in melon.     

Fusarium incarnatum is associated with postharvest fruit rot of muskmelon (Cucumis melo)

Postharvest fruit rot was observed on muskmelon (Cucumis melo) on market shelves at a melon farm in the city of Hatyai, Songkhla, Thailand. Diseased muskmelon fruit displayed cotton-like mycelia on wounds and had brown to dark brown internal tissue. Based on morphological characteristics and molecular analysis of internal transcribed spacer (ITS) and translation elongation factor 1-α (TEF1-α) DNA sequences, the fungal pathogen was identified as Fusarium incarnatum. This species belongs to the Fusarium incarnatum-equiseti species complex (FIESC). A pathogenicity test was conducted to verify Koch's postulates, and F. incarnatum was observed to cause fruit rot on muskmelon; symptoms of the disease were similar to those seen in the field. However, only artificially wounded melons became infected, suggesting that F. incarnatum is an obligate wound-infecting pathogen. To our knowledge, this is the first report of F. incarnatum causing fruit rot of muskmelon in Thailand.     

Cross-pathogenicity between Formae Speciales of Fusarium oxysporum, the Pathogens of Cucumber and Melon

The population structure of Fusarium oxysporum f. sp. cucumerinum was studied using the vegetative compatibility grouping (VCG) approach. All 37 of the examined isolates from Israel were assigned to VCG 0180, the major VCG found in North America and the Mediterranean region. Approximately two‐thirds of the tested isolates were pathogenic to both cucumber and melon, but cumulatively they were more aggressive on cucumber, their major host, than on melon. Disease symptoms on melon plants were less destructive and often expressed as growth retardation. Melon cultivars differing in Fom genes for resistance to F. oxysporum f. sp. melonis were inoculated with three isolates of F. oxysporum f. sp. cucumerinum. Results showed that Fom genes do not confer resistance to F. oxysporum f. sp. cucumerinum, although different horticultural types may respond differently to this pathogen. The reciprocal inoculation of F. oxysporum f. sp. melonis on cucumber, using four physiological races, did not result in disease symptoms or growth retardation. It is concluded that cucumerinum and melonis should remain two distinct formae speciales.     

Gaps and perspectives of pathotype and race determination in <Emphasis Type="Italic">Golovinomyces cichoracearum</Emphasis> and <Emphasis Type="Italic">Podosphaera xanthii</Emphasis>

Golovinomyces cichoracearum and Podosphaera xanthii (family Erysiphaceae) are the most important species causing cucurbit powdery mildew (CPM), a serious disease of field and greenhouse cucurbits. Both species are highly variable in their pathogenicity and virulence, as indicated by the existence of large number of different pathotypes and races. Various independent systems of CPM pathotype and race determinations and denominations are used worldwide. CPM pathotype identification is based on intergeneric and interspecific differences in host-CPM interactions. The most commonly used set of CPM pathotype differentials includes one genotype from four species representing three agriculturally important cucurbit genera plus two genotypes from a fifth species, melon Cucumis melo L. CPM races are characterized by specialization on different cultivars or lines of one host species and have, to date, been differentiated only on melon (C. melo L.). The most frequently used set of melon differentials includes 11 genotypes that can differentiate CPM races originating from melon and other cucurbits, e.g., cucumber, Cucurbita spp., and watermelon. In this paper, we critically review the current state, gaps, and perspectives in our understanding of pathogenicity variation in these two CPM pathogens at the pathotype and race levels.     

Evaluation of Alternatives to Methyl Bromide in Melon Crops in Guatemala

The monoculture of melon in Guatemala has caused the massive appearance of plants with an analogous syndrome for the well-known disease commonly called melon collapse, or vine decline, causing significant losses in crops. Methyl bromide is commonly used to sterilize soil prior to planting in Guatemala, but it must be phased out by 2015. The objective of this study was to evaluate the technique of grafting melon onto hybrids of Cucurbita (Cucurbita maxima × Cucurbita moschata), as an alternative to using soil disinfectants (such as Metam sodium, 1,3-dichloropropene, and methyl bromide) for the control of collapse. The results suggested that both soil disinfection and grafting were not necessary in these locations, since there were no statistical differences in terms of yields between the treatments and the untreated control. Furthermore, these results demonstrate that decisions to disinfect the soil must be based on the firm identification of the causal agents, in addition to preliminary assessments of yield losses.     

Effects of Preconditioning Through Mycorrhizal Inoculation on the Control of Melon Root Rot and Vine Decline Caused by Monosporascus cannonballus

The use of inoculum of arbuscular mycorrhizal fungi (AMF) in nursery represents a promising field in horticulture because of its known benefits in terms of plant growth and bioprotection. The present work was undertaken to determine the effect of mycorrhizal inoculation with Rhizophagus irregularis in a nursery medium on the containment of melon root rot and vine decline (MRRVD) caused by the soil‐borne pathogen Monosporascus cannonballus. The percentage of mycorrhization, biomass and yield following mycorrhizal inoculation were also evaluated. Biocontrol activity was assessed in greenhouse pot experiments upon artificial inoculation of M. cannonballus and in a two‐season field experiment under production conditions in an unheated greenhouse with a history of MRRVD. On the basis of the mycorrhization parameters, the interaction appeared to be established within 30 days after inoculation. The total shoot growth in the mycorrhized plants was significantly higher when compared to the control, while the root growth was unaffected. Upon artificial inoculation of M. cannonballus, mycorrhization provided complete protection against the pathogen. Greenhouse experiments under production conditions during spring cropping season showed that pretransplanting inoculation with R. irregularis significantly decreased the severity of the disease. Also, the average fruit weight of mycorrhized plants was significantly higher than the untreated control. Nevertheless, in summer crop, the bioprotection activity of AMF failed. Present results indicate that the use of AMF in a nursery setting can contribute to the prevention of the onset of this problematic soil‐borne disease within a sustainable and integrated soil‐borne disease management.     

Effect of combination between inducer resistance chemicals and systemic fungicides on management of sweet melon downy mildew

The efficiency of some inducer resistance chemicals (IRCs) like bion, chitosan, humic acid and salicylic acid as well as the fungicides like Folu-Gold, Galben Copper, Previcure-N and Redomil Gold Mancozeb on management of sweet melon downy mildew, caused by Pseudoperonospora cubensis was evaluated in vitro and in vivo. Also, the efficiency of the alternation between the sprayed two fungicides and IRCs on management of the disease and the produced fruit yield and its total soluble solids (TSS) under field conditions were assessed. The inhibitory effect of the IRCs and the tested fungicides on sporangial germination of P. cubensis resulted in a significant reduction in the germinated sporangia. In addition, IRCs were less effective than the fungicides. Disease management revealed the same trend of the in vitro experiment when they sprayed fungicides on sweet melon plants artificially inoculated with the sporangia of the causal fungus under greenhouse conditions. Furthermore, under field conditions, spraying sweet melon plants with the two tested fungicides was the most efficient in decreasing the disease and increasing fruit yield and its TSS, to somewhat, followed by the alternation between them and the tested IRCs. In addition, IRCs treatments showed the lowest efficiency in this regard.     

Soil Suppressiveness to Fusarium Wilt of Melon, Induced by Repeated Croppings of Resistant Varieties of Melons

A field soil, artificially infested with pathogenic isolates of Fusarium oxysporum f. sp. melonis was continuously used for screening resistant varieties of melon to Fusarium wilt. After 9–10 years of continuous cropping with resistant varieties, the soil had developed induced suppressiveness. Seven to 9 experimental replantings of the induced suppressive soil with the susceptible cultivar of melon, ‘Ein-Dor', nullified its suppressiveness. This was expressed by 90 % disease incidence. Only 2 replantings were required to obtain the same disease incidence in an adjacent field of a conducive soil. Nonpathogenic isolates of F. oxysporum, isolated from the rhizospheres of melon seedlings, induced various degrees of soil suppressiveness when added to soil at various ratios to the pathogenic isolate.     

Five independent loci each control monogenic resistance to gummy stem blight in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Inheritance and segregation analysis demonstrated that five independent genes in melon confer monogenic resistance to foliar infection by the fungal pathogen Didymella bryoniae, resulting in the disease known as gummy stem blight (GSB). In this study, two new monogenic sources of GSB resistance were characterized. Resistance in Cucumis melo PI 482398 was monogenic dominant based on segregation analysis of F₁, F₂ and backcross populations, while resistance in C. melo PI 482399 showed monogenic recessive inheritance. Four accessions, PI 482398, PI 157082, PI 511890, and PI 140471, each previously known to carry monogenic dominant resistance to GSB, were intercrossed to determine genetic relationships among these resistance sources. Recovery of susceptible individuals in F₂ populations confirmed that these accessions possess different resistance genes. Resistance loci were designated Gsb-1 (formerly Mc, monogenic dominant resistance from PI 140471), Gsb-2 (monogenic dominant resistance from PI 157082), Gsb-3 (monogenic dominant resistance from PI 511890), Gsb-4 (monogenic dominant resistance from PI 482398) and gsb-5 (monogenic recessive resistance from PI 482399).Communicated by J. Dvorak