Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

<Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a rhizospheric, Gram positive, rod shaped, yellow pigmented bacterium isolated from an apple orchard rhizospheric soil, on nutrient agar plates incubated at 4°C. The species level identification was arrived on the basis of 16S rRNA gene sequencing. The sequence showed 98% similarity with sequences of E. acetylicum available in the public domain. The strain was positive for siderophore and HCN production. In separate invitro assays it was found to inhibit the growth and development of Rhizoctonia solani, Sclerotium rolfsii, Pythium and Fusarium oxysporum. The volatile compound produced by the bacterium was found to be the most potent in inhibiting the hyphal development of R. solani, S. rolfsii, Pythium and F. oxysporum by 45.55, 41.38, 28.92 and 39.74% respectively. Commonly observed deformities caused by the diffusible and volatile compounds produced by the bacterium included hyphal inhibition, constriction and deformation. Under pot culture conditions the bacterium improved the germination and early growth parameters of pea (Pisum sativum) in the presence of R. solani and S. rolfsii.     

Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi.

Phytophthora cinnamomi, a member of the Pythiacease, does not synthesize sterols. Small amounts of squalene, but no squalene epoxide or sterol, were isolated from the dried mycelium of this fungus after growth in sterol-free medium. The dried mycelium of Rhizoctonia solani, a sterol-synthesizing fungus grown under the same conditions, contained small amounts of squalene and squalene epoxide and large amounts of ergosterol. When the two organisms were grown in the presence of [14C]acetate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi mycelium, whereas labelled geraniol, farnesol, squalene, squalene epoxide and ergosterol were recovered from the R. solani mycelium. Similar results were obtained when the organisms were incubated in the presence of [2(-14)C]mevalonate; in this case, labelled lanosterol was also detected in the R. solani mycelium. Both organisms, when incubated in the presence of unlabelled squalene, squalene epoxide or lanosterol, incorporated these compounds into their mycelia; however, only the R. solani mycelium was able to convert these substrates into products further along the sterol pathway. It appears that squalene is the terminal compound in the sterol biosynthetic pathway of P. cinnamomi.     

Relationship between temperature optima and secreted protease activities of three Pythium species and pathogenicity toward plant and animal hosts

The in vitro physiological characteristics of three species of Pythium (oomycetes) that utilize different food sources were compared with their ecological activities: P. insidiosum is a pathogen of mammals (including humans), P. graminicola infects the roots of graminaceous hosts, and P. grandisporangium is an enigmatic water mold isolated from mangrove leaves and marine algae. P. insidiosum and P. graminicola showed peak growth rates at 37 °C before complete inhibition of growth at 40 °C; P. grandisporangium grew fastest at 22 °C. Differences between the invasive pressures exerted by the hyphae of these microorganisms were not considered significant in relation to the substrates colonized by these water molds. All three species showed substantial secreted protease activity, producing three or more serine proteases with weights ranging from 24-38 kDa. Fastest growth rates were supported when collagen was supplied as the sole carbon source, and none of the species were able to grow on purified plant cell wall polysaccharides. The growth and nutritional characteristics of P. graminicola and P. grandisporangium bear little obvious relationship to the ecological niches that they inhabit. This highlights the caution necessary in extrapolating from laboratory analyses to the natural environment, and points to the potential importance of ecological opportunity in determining the host range and food source of certain microorganisms.     

The effect of sterols on the metabolism of Pythium species

Summary The growth of several Pythium species is increased between 65 and 100% if cholesterol is added to the growth medium. The optimum concentration is 15 mcg per ml. Mycelium of Pythium ultimum, in which cholesterol is present, incorporates glucose-U-¹⁴C and releases ¹⁴CO₂ at a faster rate than the corresponding sterol free mycelium. In sterol containing cells, more ¹⁴CO₂ is produced from a given amount of absorbed glucose-U-¹⁴C than in sterol free cells, there is thus in sterol containing hyphae a higher level of energy production. This condition can account for the increase in growth due to cholesterol. Only if sterols are present in the cellular membranes of Pythium species is the optimum synthetic capacity reached.     

Evidence from cell-free systems for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi.

Cell-free preparations of both Rhizoctonia solani, a sterol-synthesizing fungus, and Phytophthora cinnamomi, a non-sterol-synthesizing fungus, incubated in the presence of [2(-14)C]mevalonate and iodacetamide, converted the mevalonate into labelled mevalonate 5-phosphate, mevalonate 5-pyrophosphate and isopentenyl pyrophosphate. In the absence of iodoacetamide, but under anaerobic conditions, the same preparations converted the mevalonate into labelled geraniol, farnesol and squalene, the first two compounds presumably as their pyrophosphates. When cell-free preparations of both organisms were incubated aerobically in the presence of [1(-14)C]isopentenyl pyrophosphate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi reaction mixture, whereas labelled geraniol, farnesol, squalene, squalene epoxide, lanosterol and ergosterol were present in the R. solani reaction mixture. When these same preparations were incubated in the presence of 14C-labelled squalene, labelled squalene epoxide, lanosterol and ergosterol were recovered from the R. solani reaction mixture. In contrast, the P. cinnamomi preparation was unable to convert the squalene into products further along the sterol pathway; instead, a portion of the labelled squalene was converted into water-soluble products, indicating the possible existence of a squalene-degradation process in this organism. It appears that the block in the sterol biosynthetic pathway of P. cinnamomi occurs at the level of squalene epoxidation.     

Development of Multiplex PCR to Detect Five Pythium Species Related to Turfgrass Diseases

The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species‐specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.     

Isolation,identification, carbon utilization profile and control of Pythium graminicola,the causal agent of chilli damping-off

Postemergence damping-off of chilli caused by Pythium spp. is a common and serious problem in large chilli growing areas of India under the moist conditions that generally prevails during the sowing period. Therefore, in order to better understand this disease, an isolate belonging to the genus Pythium (Pythiales) was isolated from the infected chilli (Capsicum annuum L.) plant root parts collected from the fields of Chandauli district, Uttar Pradesh, India. Based on the congruence of cultural, morphological, cardinal growth rate and the sequence data analysis, the isolate was identified as Pythium graminicola. The molecular phylogenetic analysis based on ITS-rDNA sequences clustered the isolate with representative sequences for P. graminicola from GenBank in the Pythium clade. The isolate carbon utilization profiles were characterized using Biolog FF MicroPlate method. The results revealed that the isolate used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Moreover, an in vitro colony growth inhibition assay was performed to determine the influence of chemical (fungicides) and biological (bacteria and fungi) antagonists over the pathogen using the poison plate and dual culture method, respectively. Overall, the results revealed that the presence of aggressive broad range biocontrol agents can be used as an effective environmentally friendly approach for management and control of damping-off in production systems. The antagonist can serve as a bio-efficient and eco-friendly alternative to synthetic fungicides for the development of an effective integrated pest management (IPM) system and obtaining higher yields.     

Rhizotron studies on Zea mays L. to evaluate biocontrol activity of Bacillus subtilis

The effect of Bacillus as a biocontrol agent against some root-rot fungi was tested using maize (Zea mays L.) in rhizotrons placed in a growth chamber with relative humidity 60% with a 12 h photoperiod and day and night temperatures of 24 and 18°C respectively. Rhizoctonia solani Kühn caused pre-emergence damping-off in the untreated maize seeds showing weak and soft roots as observed through the perspex rhizotrons. Image analysis was used to quantify the effects of Bacillus treatment on seedlings infected with Pythium sp. Bacillus B77 and B81 were most effective in the control of the pathogen, R. solani which achieved a biocontrol activity of 24 and 35% respectively with regard to shoot dry biomass while B81 achieved 48% biocontrol with reference to root dry biomass. There was no effect on the root area. For root dry biomass, B81, B69, B11 and B77 showed higher biocontrol activity in comparison to the control. Pythium sp. caused pre- and post emergence damping- off in the untreated seeds. Root rot of the maize seedlings caused by Pythium sp. was slightly controlled by Bacillus B69 and B81 which achieved biocontrol activity of 18 and 11% respectively. For the biocontrol of Fusarium solani, Bacillus B77, B69, B81 achieved biocontrol activity of 50, 48 and 33% respectively with reference to root dry biomass.     

The incorporation of acetate by the chemoautotroph Thiobacillus neapolitanus strain C

Summary Cultures of Thiobacillus neapolitanus strain C assimilate ¹⁴C-labelled acetate and aspartate. Both carbon atoms of acetate are incorporated, and 25% of the cell carbon can arise from acetate. Aspartate-¹⁴C contributes 4–5% of the cell carbon, and is found in pyrimidines and in protein as aspartate and its related amino acids. Acetate-¹⁴C contributes to lipid, glutamate, arginine, proline and leucine, but not to aspartate. Acetate assimilation by washed organisms requires carbon dioxide and energy from thiosulphate oxidation. Degradation of ¹⁴C-glutamic acid from acetate-¹⁴C-labelled bacteria; the accumulation of ¹⁴C-citrate in the presence of fluoroacetate and [¹⁴C] acetate; short-term kinetic experiments on acetate-¹⁴C turnover; and the demonstration of citrate synthesis by cell-free extracts all indicate glutamate synthesis from -ketoglutarate formed by reactions of the tricarboxylic acid cycle. The cycle is believed to be incomplete, probably not proceeding further than -ketoglutarate, and functions as a glutamate-synthesising system, using oxaloacetate derived solely from carbon dioxide fixation. Malate synthase (and the glyoxylate cycle) appear to be insignificant in the metabolism, but extracts did form citramalate from acetate and pyruvate.     

Effect of the high polycyclic aromatic hydrocarbon, benzo[a]pyrene, on the lipid content of Fusarium solani

The purpose of the present paper was to study the effect of the high polycyclic aromatic hydrocarbon (PAH), benzo[a]pyrene, on the lipid [fatty acid (FA) and sterol] composition and content of the fungi Fusarium solani and F. oxysporum, respectively recognized as good and poor PAH degraders. The major FAs and the major sterol that characterized the tested Fusarium strains were C16:0, C18:1, C18:2, and ergosterol. Lipid profiles of F. solani remained unchanged with the addition of benzo[a]pyrene in the culture media at all concentrations and duration of treatment. However, in the presence of benzo[a]pyrene, significant decreases in FA content, which reached 18 % in young cultures and 28 % in mature colonies, were registered. Similarly, the sterol content of F. solani was reduced by 27 % in the presence of benzo[a]pyrene. In contrast, no modification in lipid profile and lipid content were observed with F. oxysporum, a strain recognized as a low benzo[a]pyrene degrader.     

Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I₁₇ produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I₁₇ which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Survival and recovery of Phytophthora capsici and oomycetes in tailwater and soil from vegetable fields in Florida

A current trend in Florida agriculture to conserve water is to irrigate with surface runoff water (tailwater) recovered in retention ponds and canals. Water filtration and lemon leaf baiting recovered Phytophthora capsici and other plant pathogenic Oomycetes in runoff water from ponds and canals. A total of 196 isolates of Phytophthora spp. and 471 isolates of Pythium spp. were recovered. Phytophthora spp. included P. capsici, P. cinnamomi, P. lateralis, P. nicotianae, P. citricola, P. cryptogea and P. erythroseptica. Species of Pythium were P. aphanidermatum, P. catenulatum, P. helicoides, P. irregulare, P. myriotylum, and Pythium‘group F’. Isolates of P. aphanidermatum, P. irregulare, P. myriotylum, and Pythium‘group F’ were pathogenic on pepper and tomato. Recovery of P. capsici propagules was related to soil moisture‐holding capacity and time interval but not temperature. Recovery of P. capsici propagules at 100% soil moisture‐holding capacity and 30° C was 57 days. In tailwater, recovery of propagules of P. capsici was 63 days at 24°C to 25°C. The potential exists to reintroduce and disseminate species of Phytophthora and Pythium when using tailwater for irrigation or other practices.     

Growth and Protein Content in Colletotrichum circinans, Fusarium solani and Rhizoctonia solani in Liquid Culture

The phytopathogenic fungi Colletotrichum circinans, Fusarium solani, and Rhizoctonia solani were incubated in aerated (0, 0.5, 1 dm³ min^–1) potato dextrose broth (PDB) or Czapek-Dox broth (CDB), under 0-, 12- or 24-h photoperiods. Greater dry mass was produced in PDB. Higher air flows improved dry mass of F. solani and R. solani. The 24-h photoperiod improved F. solani dry mass. Except for F. solani, which was not affected, incubation in PDB increased protein content. The no air treatment increased protein content in F. solani, 0.5 dm³ min^–1 produced the highest protein content in R. solani, but air flow-rate did not affect C. circinans. Incubation in the dark produced the lowest protein content in C. circinans, the highest under the 24-h photoperiod for R. solani, and photoperiod did not affect protein content in F. solani. Protein content in R. solani, incubated in CDB, was lowest at the 0 dm³ min^–1 air flow at all photoperiods. Molecular masses of most proteins were under 30 kDa, and numbers of bands in SDS-PAGE gels varied with air flow. In CDB, especially under 12- or 24-h photoperiods, proteins in F. solani were between 1.6 and 310 kDa. For R. solani in PDB, at 0.5 dm³ min^–1 air flow and 12-h light, proteins were between 6 and 81 kDa.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

Construction of squalene-accumulatingSaccharomyces cerevisiae mutants by gene disruption through homologous recombination

Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying theLEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.