Effect of Indole-3-Acetic Acid (IAA) Produced by Pseudomonas aeruginosa in Suppression of Charcoal Rot Disease of Chickpea

The production of indole-3-acetic acid (IAA), by rhizobacteria, has been associated with plant growth promotion, especially root initiation and elongation. Isolate TO3 selected from 103 fluorescent pseudomonads, identified as Pseudomonas aeruginosa, showed maximum production of IAA. Isolate TO3 having biocontrol activity against Macrophomina phaseolina also showed production of siderophore and HCN was used to screen the role of bacterial IAA in reducing the level of charcoal rot disease occurrence in chickpea. Four IAA defective stable mutants of isolate TO3 having biocontrol activity against M. phaseolina were developed through 5-bromouracil mutagenesis. Mutant TO₅₂ showed 76.47% reduction in production of IAA. Standard IAA was used in similar concentration as present in cell-free culture supernatant of wild isolate TO3 and its mutant TO₅₂. The in vitro and in vivo study showed that IAA-defective mutant TO₅₂ caused reduced biocontrol and plant growth promotory activity than wild isolate TO3. Standard IAA showed comparable biocontrol activity to the culture supernatant. To some extent better biocontrol and growth promotory activity in supernatant than standard IAA indicates the synergistic role of siderophore and HCN. The study clearly reports the role of bacterial IAA in suppression of charcoal rot disease of chickpea.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

Diversity and activity of biosurfactant-producing Pseudomonas in the rhizosphere of black pepper in Vietnam

Aims: Phytophthora capsici is a major pathogen of black pepper and zoospores play an important role in the infection process. Fluorescent pseudomonads that produce biosurfactants with zoosporicidal activities were isolated from the black pepper rhizosphere in Vietnam, and their genotypic diversity and potential to control Phy. capsici root rot was determined. Methods: Biosurfactant‐producing pseudomonads were genotypically and biochemically characterized by BOX‐polymerase chain reaction (PCR), 16S‐rDNA sequencing, reverse‐phase‐high‐performance liquid chromatography and liquid chromatography‐masss spectrometry analyses. Results: Biosurfactant‐producing fluorescent pseudomonads make up c. 1.3% of the culturable Pseudomonas population in the rhizosphere of black pepper. Although BOX‐PCR revealed substantial genotypic diversity, the isolates were shown to produce the same biosurfactants and were all identified as Pseudomonas putida. When applied to black pepper stem cuttings, several of the biosurfactant‐producing strains provided significant disease control. In absence of the disease, several of the bacterial strains promoted shoot and root growth of black pepper stem cuttings. Conclusions: Biosurfactant‐producing pseudomonads indigenous to the rhizosphere of black pepper plants are genotypically diverse and provide a novel resource for the control of Phy. capsici root rot and growth promotion of black pepper stem cuttings. Significance and Impact of the Study: The results of this study provide a strong basis for further development of supplementary strategies with antagonistic bacteria to control foot and root rot of black pepper and to promote plant growth.     

Rhamnolipid–Biosurfactant Permeabilizing Effects on Gram-Positive and Gram-Negative Bacterial Strains

The potential of biosurfactant PS to permeabilize bacterial cells of Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis on growing (in vivo) and resting (in vitro) cells was studied. Biosurfactant was shown to have a neutral or detrimental effect on the growth of Gram-positive strains, and this was dependent on the surfactant concentration. The growth of Gram-negative strains was not influenced by the presence of biosurfactant in the media. Cell permeabilization with biosurfactant PS was shown to be more effective with B. subtilis resting cells than with Pseudomonas aeruginosa. Scanning-electron microscopy observations showed that the biosurfactant PS did not exert a disruptive action on resting cells such that it was detrimental to the effect on growing cells of B. subtilis. Low critical micelle concentrations, tender action on nongrowing cells, and neutral effects on the growth of microbial strains at low surfactant concentrations make biosurfactant PS a potential candidate for application in different industrial fields, in environmental bioremediation, and in biomedicine.     

Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola, Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.     

Metabolites Composition of <i>Bacillus subtilis HussainT-AMU</i> Determined by LC-MS and Their Effect on Fusarium Dry Rot of Potato Seed Tuber

Fusarium dry rot is considered to be the most critical soilborne and postharvest disease that damages potato tubers worldwide when they are stored for a long time. This study was performed to demonstrate the effect of crude extract, culture filtrate, and cell suspension obtained from the bacterium Bacillus subtilis HussainT-AMU, on the net house and field. From oil-contaminated soil, through the serial dilution method, biosurfactant bacteria were isolated on nutrient agar medium. To isolate and screen the prospective biosurfactant strain, various biosurfactant screening methods were used. Standard protocols were carried out for morphological, molecular, and chemical characterization. The disease incidences were significantly decreased under net house and field conditions after treatment with biosurfactant extract (20%, 70%), culture filtrate (23%, 49%), and bacterial cell suspension (46%, 27%), respectively. These results indicate a significant effect of B. subtilis HussainT-AMU against Fusarium oxysporum. The characterization of the partially purified extract by gas-chromatography indicated the existence of a high amount of methyl ester fatty acids. The quantitative analysis determined the presence of lipopeptide-surfactin, fengycin, and iturin in the biosurfactant extract. Scanning electron microscopy revealed that morphological variation was revealed in the structure of stomata size in leaves and in the mycelia of the pathogen that was investigated. This promising biocontrol agent can be a plausible option for further research and can also be used for the production of green biomolecules at an additional industrial level.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida antarctica (sp. SY16) required avegetable oil as the carbon source to produce a biosurfactant, mannosylerythritol lipid (MEL-SY16). Biosurfactant production was 31 g l^–1 after 7 days in a batch culture and was not growth associated. In a two-stage culture, glycerol and oleic acid were used as an initial and a feeding carbon source, respectively, and 41 g l^–1 biosurfactant was produced after 8 days.     

Degradation of hydrocarbons and biosurfactant production by Pseudomonas sp. strain LP1

Pseudomonas sp. strain LP1, an organism isolated on the basis of its ability to grow on pyrene, was assayed for its degradative and biosurfactant production potentials when growing on crude, diesel and engine oils. The isolate exhibited specific growth rate and doubling time of 0.304 days⁻¹ and 2.28 days, respectively on crude oil (Escravos Light). The corresponding values on diesel were 0.233 days⁻¹ and 2.97 days, while on engine oil, were 0.122 days⁻¹ and 5.71 days. The organism did not show significant biosurfactant production towards crude oil and diesel, but readily produced biosurfactant on engine oil. The highest Emulsification index (E₂₄) value for the biosurfactant produced by LP1 on engine oil was 80.33 ± 1.20, on day 8 of incubation. Biosurfactant production was growth-associated. The surface-active compound which exhibited zero saline tolerance had its optimal activity at 50°C and pH 2.0.     

Isolation and characterization of a biosurfactant producing strain, <Emphasis Type="Italic">Brevibacilis brevis</Emphasis> HOB1

Biosurfactant-producing bacteria were isolated from the production water of an oil field. Isolates were screened for biosurfactant production using surface tension test. The highest reduction of surface tension was achieved with a bacterial strain which was identified by 16S rRNA gene sequencing as Brevibacilis brevis HOB1. It has been investigated using different carbon and nitrogen sources. It showed that the strain was able to grow and reduce the surface tension of the broth to 29 mN/m on commercial sugar and maltose, and to 32 mN/m on glucose after 72 h of growth. The maximum amount of biosurfactant was obtained when nitrate ions were supplied as nitrogen source. Biosurfactant produced by Brevibacilis brevis HOB1 was confirmed as a lipopeptide class of biosurfactant using TLC test and mass spectra. Lipopeptide isoforms were isolated from cell-free supernatants by acid-precipitation followed by one step of chromatographic separation on solid-phase ODS C18 column. The separation was confirmed by HPLC and ESI Q-TOF MS spectroscopy. Comparing the mass data obtained and the mass numbers reported for the lipopeptide complexes from other strains, it can be concluded that the major lipopeptide product of Brevibacilis brevis HOB1 is the surfactin isoform. This lipopeptide showed strong antibacterial and antifungal activity. It is a candidate for the biocontrol of pathogens in agriculture and other industries.     

Isolation of biosurfactant producers, optimization and properties of biosurfactant produced by Acinetobacter sp. from petroleum-contaminated soil

Aims: To screen and identify biosurfactant producers from petroleum‐contaminated soil; to use response surface methodology (RSM) for medium optimization to enhance biosurfactant production; and to study the properties of the newly obtained biosurfactant towards pH, temperature and salinity. Methods and Results: We successfully isolated three biosurfactant producers from petroleum‐contaminated soil and identified them through 16S rRNA sequence analysis, which exhibit the highest similarities to Acinetobacter beijerinckii (100%), Kocuria marina (99%) and Kineococcus marinus (99%), respectively. A quadratic response model was constructed through RSM designs, leading to a 57·5% increase of the growth‐associated biosurfactant production by Acinetobacter sp. YC‐X 2 with an optimized medium: beef extract 3·12 g l^?1; peptone 20·87 g l^?1; NaCl 1·04 g l^?1; and n‐hexadecane 1·86 g l^?1. Biosurfactant produced by Acinetobacter sp. YC‐X 2 retained its properties during exposure to a wide range of pH values (5–11), high temperatures (up to 121°C) and high salinities [up to 18% (w/v) Na⁺ and Ca²⁺], which was more sensitive to Ca²⁺ than Na⁺. Conclusions: Two novel biosurfactant producers were isolated from petroleum‐contaminated soil. Biosurfactant from Acinetobacter sp. YC‐X 2 has good properties to a wide range of pH, high temperature and high salinity, and its production was optimized successfully through RSM. Significance and Impact of the Study: The fact, an increasing demand of high‐quality surfactants and the lack of cost‐competitive bioprocesses of biosurfactants for commercial utilization, motivates researchers to develop cost‐effective strategies for biosurfactant production through isolating new biosurfactant producers with special surface‐active properties and optimizing their cultural conditions. Two novel biosurfactant producers in this study will widen our knowledge about this kind of micro‐organism. This work is the first application of RSM designs for cultural optimization of biosurfactant produced by Acinetobacter genus and the first report that biosurfactant may be more sensitive to Ca²⁺ than Na⁺.     

Competition is the mechanism of biocontrol of brown rot in stone fruit by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

The mechanism of biocontrol of brown rot in stone fruit by Penicillium frequentans Westling (Pf909) was investigated using in vitro and in vivo growth assays and a benomyl-resistant strain of Monilinia fructicola (G Winter) Honey (Mf3C). For the in vitro assays, Pf909 and Mf3C conidia were suspended in Czapek-Dox broth, which was amended or not amended with a skin extract of mature peaches. The growth and germination of Pf909 and Mf3C conidia were determined by counting the number of colony-forming units on potato dextrose agar plates, which were amended or not amended with 0.5 g ml^?1 benomyl. In some of the assays, germinated Pf909 conidia were used before their exposure to Mf3C conidia. For the in vivo assays, healthy cherries were inoculated with Mf3C conidia before and after applying Pf909 conidia on the cherry surface and the incidence of brown rot was recorded for seven days. Since we found that Pf909 conidia compete with Mf3C conidia for space and nutrients in the different assays, we concluded that competition is the probable primary mechanism of biocontrol of Pf909.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

Production and characterization of lipopeptide biosurfactant by a sponge-associated marine actinomycetes <Emphasis Type="Italic">Nocardiopsis alba</Emphasis> MSA10

A sponge-associated marine actinomycetes Nocardiopsis alba MSA10 was screened and evaluated for the production of biosurfactant. Biosurfactant production was confirmed by conventional screening methods including hemolytic activity, drop collapsing test, oil displacement method, lipase production and emulsification index. The active compound was extracted with three solvents including ethyl acetate, diethyl ether and dichloromethane. The diethyl ether extract was fractionated by TLC and semi-preparative HPLC to isolate the pure compound. In TLC, a single discrete spot was obtained with the R _f 0.60 and it was extrapolated as valine. Based on the chemical characterization, the active compound was partially confirmed as lipopeptide. The optimum production was attained at pH 7, temperature 30°C, and 1% salinity with glucose and peptone supplementation as carbon and nitrogen sources, respectively. Considering the biosurfactant production potential of N. alba, the strain could be developed for large-scale production of lipopeptide biosurfactant.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Renewable Resources

This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.     

Streptomyces sp. S160: a potential antagonist against chickpea charcoal root rot caused by Macrophomina phaseolina (Tassi) Goid

Biological control of charcoal root rot disease caused by Macrophomina phaseolina in chickpea was studied by using Streptomyces sp. S160. This biocontrol agent (BCA) inhibited the mycelial growth of M. phaseolina by 50 % in vitro and significantly reduced charcoal rot incidence in the greenhouse by 33.3 %. The greenhouse experiment revealed that seed treatment along with soil application supported the highest germination (88.6 %), vigor index (7326.91) and reduced root rot incidence (12.5 %) in comparison to seed treatment and soil application alone. BCA enhanced the growth and helped in inducing resistance against charcoal rot disease of chickpea caused by M. phaseolina by increasing activity of defense-related enzymes in chickpea plants, leading to the synthesis of defense chemicals in plants. BCA (Streptomyces sp. S160) was also characterized and identified by using polyphasic approaches including 16S rDNA sequencing.     

Improved germination efficiency of Salicornia ramosissima seeds inoculated with Bacillus aryabhattai SP1016‐20

Saline agriculture and the crop cultivation of halophytes represent an alternative for the reclamation of salinized soils and for the management of irrigation water. Halotolerant plant growth promoting bacteria with biocontrol effect, as an alternative to commercial fungicides, may contribute to improve crop productivity while mitigating saline stress effects. The objective of this work was to isolate autochthonous rhizobacteria with biocontrol features, to be used as germination enhancers and plant‐growth promoting agents in the crop cultivation of Salicornia ramosissima. A set of isolates obtained from the rhizosphere of S. ramosissima was characterised in terms of Gram, motility, salt tolerance and biocontrol traits (hydrogen cyanide production, antifungal activity and production of extracellular lipases and proteases). One Gram‐positive motile isolate that tested positive for all biocontrol traits was identified by 16S rRNA gene sequencing as Bacillus aryabhattai. The inoculation of S. ramosissima seeds with B. aryabhattai SP1016‐20 reduced the negative effect of salinity on the germination efficiency. At the highest tested salinity (30 g/L NaCl) the final germination efficiency of inoculated seeds doubled in relation to non‐inoculated controls. Although the mechanisms involved in the biocontrol effect were not defined in the current work, the results highlight the potential of Bacillus aryabhattai SP1016‐20 as a plant‐growth promoting agent for the crop cultivation of Salicornia and contribute to the strengthening of the scientific basis of biosaline agriculture and plant growth promoting rhizobacteria‐assisted crop cultivation of halophytes in saline soils and estuarine sediments.     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Rhizobacterial bioformulation for the effective management of Macrophomina root rot in mungbean

Abstract

Fluorescent pseudomonads based bioformulation was evaluated for their ability to control Macrophomina root rot disease in mungbean (Vigna mungo). P. fluorescens isolate Pf1 showed the maximum inhibition in mycelial growth of Macrophomina phaseolina under in vitro conditions. Bioformulation of Pf1 with chitin was effective in reducing the root rot incidence in green gram both under glasshouse and field conditions. The rhizosphere colonization of P. fluorescens was observed appreciable with the green gram plants. However, Pf1 amended with chitin colonized effectively. Furthermore, the induction of defence-related enzymes and chemicals in plants by Pf1 amended with or without chitin and neem were tested. Increased accumulation of defence enzymes viz., phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), chitinase, β-1,3-glucanse and phenolics were observed in Pf1 bioformulation amended with chitin, pre-treated plants challenge inoculated with M. phaseolina under glasshouse conditions. The present study reveals that in addition to direct antagonism and plant-growth promotion, PGPR strains amended with chitin bioformulation induced defence-related enzymes and pathogenesis related (PR) proteins which collectively enhance the resistance in green gram against the infection of M. phaseolina.     

Potential of endophytic Streptomyces spp. for biocontrol of Fusarium root rot disease and growth promotion of tomato seedlings

Sixteen endophytic actinobacteria isolated from roots of native plants were evaluated for their antagonistic potential against soil-borne phytopathogenic fungi. Among them, three strong antagonistic isolates were selected and characterised for in vitro plant-growth-promoting and biocontrol traits, including production of hydrogen cyanide, indole-3-acetic acid and siderophores, chitinase and β-1,3-glucanase activities, and inorganic phosphate solubilisation. In all trials, the strain Streptomyces sp. SNL2 revealed promising features. The selected actinobacteria were investigated for the biocontrol of Fusarium oxysporum f. sp. radicis lycopersici and for growth promotion of tomato (Solanum lycopersicum L. cv. Aïcha) seedlings in autoclaved and non-autoclaved soils. All seed-bacterisation treatments significantly reduced the root rot incidence compared to a positive control (with infested soil), and the isolate SNL2 exhibiting the highest protective activity. It reduced the disease incidence from 88.5% to 13.2%, whereas chemical seed treatment with Thiram® provided 14.6% disease incidence. Furthermore, isolate SNL2 resulted in significant increases in the dry weight, shoot and root length of seedlings. 16S rDNA sequence analysis showed that isolate SNL2 was related to Streptomyces asterosporus NRRL B-24328^T (99.52% of similarity). Its interesting biocontrol potential and growth enhancement of tomato seedlings open up attractive uses of the strain SNL2 in crop improvement.     

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.