m-Hydroxyphenylacetic and m-Methoxyphenylacetic Acids of Rhizoctonia solani: Their Effect on Specific Root-Nodule Activity and Histopathology in Soybean

m-Methoxyphenylacetic acid (m-OMePAA), a derivative of m-hydroxyphenylacetic acid (m-OHPAA), having the same chemical composition as that phytotoxic compound produced in culture by Rhizoctonia solani, a fungal pathogen of soybean, reduced growth and symbiotic N₂-fixation activity of ‘Tracy’ soybeans in soil: perlite at 1.5 × 10^-4 M, the lowest concentration used. At twice this concentration m-OMePAA was strongly teratogenic and caused root hypertrophy and root fasciation. At 1.2 × 10^-3 M, m-OMePAA nearly suppressed seed germination. m-OMePAA at the minimum concentration used and equivalent concentrations of the culture filtrate fraction (m-OHPAA and m-OMePAA) caused cytopathological and histopathological disturbances in the nodule central tissue, extrusion of the nucleoli, and abnormal nuclci. The data indicate that these phytotoxic compounds of R. solani are involved in nodule impairment and reduced N₂-fixation in soybean.     

Plant growth promotion and suppression of charcoal‐rot fungus (Macrophomina phaseolina) in velvet bean (Mucuna pruriens L.) by root nodule bacteria

Root‐nodulating bacteria are intimate associates of legumes. From a pool of rhizobia isolated from root nodules of Mucuna pruriens (Velvet bean/Kaunch), RMP66 and BMP17 were found to be capable of promoting siderophore and IAA production and phosphate solubilization (insoluble tri‐calcium). Both symbionts were studied further to determine their abilities to promote plant growth and to control root‐rot in Mucuna pruriens caused by the pathogenic plant fungus Macrophomina phaseolina. RMP66 and BMP17 were selected based on their excellent inhibitory activities against M. phaseolina (by 78% and 71%, respectively) in dual culture and in agar‐well assays using cell‐free culture filtrate (CFCF) (by 76% and 62%, respectively). Both strains inhibited fungal growth to a greater extent in iron‐deficient medium (51% and 69%) than in iron‐supplemented medium (37% and 0%), respectively. CFCFs of RMP66 and BMP17 obtained from Pikovskaya's broth and tryptophan‐amended YEM broth inhibited fungal growth by 80%‐55% and 70%‐43%, respectively, and were identified as Sinorhizobium meliloti RMP66 and Bradyrhizobium diazoefficiens BMP17 by 16S rDNA sequencing. Centrifuged and pelleted cells harvested from exponentially grown cultures of S. meliloti RMP66 and B. diazoefficiens BMP17 were used to bacterize seeds of M. pruriens, which then showed enhanced seed germination (by up to 17% and 12%, respectively), and subsequent increases in other plant growth parameters in field trials. Considerable increases in seedling vigour indices (62%: 53% and 110%: 130%) and biomass (8%: 13% and 25%: 28%) were also observed for bacterial treatments. Tn5‐mediated antibiotic‐resistant marker strains showed enhanced nodule occupancy by up to 72% and 68%, respectively. This study describes a multifunctional legume nodule rhizobia that could be utilized in multicropping systems under different agroclimatic conditions as a bioinoculant and alternative to fertilizers.     

Experimental Transmission of Plum Pox Virus (PPV) to Prunus Mahaleb and Prunus avium

Sharka caused by plum pox virus (PPV) is a disease spread in France since 1970, and causing severe damages essentially on apricot but also on plums and peach. Cherry is generally considered as not infected by PPV. Experimental transmissions by chip budding or aphids allowed to show that 3 isolates of PPV can multiply inside three cherry rootstocks (P. Mahaleb cv.‘SL 64′, P. avium cv.‘F 12-1′, and P. avium*P. pseudocerasus cv. ‘Colt') (Tables 1 and 2). But generally, the virus remained localized to the infection site and disappeared quickly (Table 3). Typical symptoms of chlorotic ringspot or vein clearing are also limited to the leaves probed by the aphids. The fact that no translocation was detected is discussed.     

Biological control of root rot fungus <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and growth enhancement of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> (Sarg.) by rhizosphere competent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BN1

Bacterial isolates having antifungal and good plant growth-promoting attributes were isolated from chir-pine (Pinus roxburghii) rhizosphere. An isolate, Bacillus subtilis BN1 exhibited strong antagonistic activity against Macrophomina phaseolina, and other phytopathogens including Fusarium oxysporum and Rhizoctonia solani. It was characterized and selected for the present studies. BN1 resulted in vacuolation, hyphal squeezing, swelling, abnormal branching and lysis of mycelia. The cell-free culture filtrate of BN1 inhibited the growth of M. phaseolina. Pot trial study resulted in statistically significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. BN1 treatment resulted in 43.6% and 93.54% increases in root and shoot dry weights respectively, as compared to control. Also, 80–85% seed viability was recorded in treatments receiving BN1 either alone or in the presence of M. phaseolina, compared to 54.5% with M. phaseolina. Bioinoculant formulation study suggested that maximum viability of bacteria was in a sawdust-based carrier. B. subtilis BN1 produced lytic enzymes, chitinase and β-1,3-glucanase, which are known to cause hyphal degradation and digestion of the cell wall component of M. phaseolina. In the presence of M. phaseolina, population of B1 was 1.5 × 10⁴ c.f.u. g⁻¹ root after one month, which increased to 4.5 × 10⁴ c.f.u. g⁻¹ root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potent biocontrol agent.     

An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.     

Inhibition of seed germination by Macrophomina phaseolina is related to phaseolinone production

The production of phaseolinone, a phytotoxic metabolite of Macrophomina phaseolina in infected Phaseolus mungo seeds grown on soil, was estimated by enzyme-linked immunosorbent assay and HPLC. The degree of inhibition of seed germination correlated well with the amount of toxin produced; 50% inhibition was observed at a toxin level of 2.1 μg g^-1 of wet tissue. A comparison of the toxin-producing ability of nine isolates of the fungus obtained from different hosts and localities showed that the strain MPK'83 produced a significantly larger amount of the toxin, both in liquid culture and in infected seeds. The virulence of the isolates was related to their ability to produce phaseolinone.     

CHROMOSOME PAIRING AND POLLEN FERTILITY IN INTERSPECIFIC HYBRIDS OF SPECIES OF PARTHENIUM (ASTERACEAE)

Meiotic analyses and pollen viability tests were performed on F, hybrids between diploid guayule (Parthenium argentatum Gray 2n = 36), P. rollinsianum Rzedowski (2n = 36), P. alpinum var. tetraneuris Barneby (2n = 36), and P. alpinum var. alpinum Nutt. (2n = 36). Parthenium chromosomes are small and karyomorphologically similar, and meiotic analysis is difficult because of chromosome clumping. However, cytogenetic studies at metaphase I indicated univalents can be seen in a lateral view of the metaphase plate. Chromosome pairing and the number of univalents varied within and between the interspecific hybrids, with an average univalent number of 1.54 for the P. rollinsianum hybrids, 2.36 for the P. alpinum var. tetraneuris hybrids, and 2.46 for the P. alpinum var. alpinum hybrids. Pollen viability tests for the parental species and the hybrids were conducted by germination of pollen grains on stigmas. The percent of viable pollen recorded for the diploid guayule hybrids with P. rollinsianum, P. alpinum var. tetraneuris, and P. alpinum var. alpinum are 21.94, 13.47, and 11.17, respectively. The degree of chromosome pairing and pollen viability is striking because there are many morphological differences between the parents. The chromosome homology of these species based on their pairing behavior allows for the design of a backcross breeding program that would permit the transfer of the desirable characteristics from these species into diploid guayule.     

An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.     

Optimization of Culturing Conditions of a Strain of Phytophthora capsici Pathogenic to Habanero Pepper (Capsicum chinense)

Phytophthora capsici is an oomycete known as the causal agent of wilting disease in Capsicum spp., which causes rotting of roots, crowns, stems, leaves and fruits. To date, little is known about the production of phytotoxic metabolites by P. capsici or their role in the infection process. As part of a project directed towards the isolation and identification of phytotoxins produced by a strain of P. capsici pathogenic to habanero pepper (Capsicum chinense), we have evaluated the effect of factors such as aeration, light and culture medium on the production of mycelium and phytotoxic metabolites by P. capsici. The results showed that culturing P. capsici in potato dextrose broth (PDB) containing habanero pepper leaf infusion, in the dark and under still conditions, results in a high production of mycelium and a high phytotoxicity of the culture filtrate, in the shortest period of time.     

A quantitative assessment of the antimicrobial activity of garlic (Allium sativum)

An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml^-1. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml^-1. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 g ml^-1. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia coli, garlic prevented the establishment of E. coli, although the final outcome of competition was not affected.L.P. Rees, S.F. Minney and N.T. Plummer are with Interprise Ltd, Baglan Bay Industrial Park, Port Talbot SA 12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.     

Antagonistic potential of native strain Streptomyces aurantiogriseus VSMGT1014 against sheath blight of rice disease

A total of 132 actinomycetes was isolated from different rice rhizosphere soils of Tamil Nadu, India, among which 57 showed antagonistic activity towards Rhizoctonia solani, which is sheath blight (ShB) pathogen of rice and other fungal pathogens such as Macrophomina phaseolina, Fusarium oxysporum, Fusarium udum and Alternaria alternata with a variable zone of inhibition. Potential actinomycete strain VSMGT1014 was identified as Streptomyces aurantiogriseus VSMGT1014 based on the morphological, physiological, biochemical and 16S rRNA sequence analysis. The strain VSMGT1014 produced lytic enzymes, secondary metabolites, siderophore, volatile substance and indole acetic acid. Crude metabolites of VSMGT1014 showed activity against R. solani at 5 µg ml^?1; however, the prominent inhibition zone was observed from 40 to 100 µg ml^?1. Reduced lesion heights observed in culture, cells-free filtrate, crude metabolites and carbendazim on challenge with pathogen in the detached leaf assay. The high content screening test clearly indicated denucleation of R. solani at 5 µg ml^?1 treatment of crude metabolite and carbendazim respectively. The results conclude that strain VSMGT1014 was found to be a potential candidate for the control of ShB of rice as a bio fungicide.     

Biostoning of denims by Penicillium occitanis (Pol6) cellulases

The Pol6 mutant of Penicillium occitanis, secreting a large quantity of cellulases, was cultivated in fermentor using a local paper pulp as an inducer substrate. A high titer of extracellular cellulase activity was reached after a fed batch process: 23 IU ml⁻¹ filter paper activity, 21 IU ml⁻¹ CMCases activity (endoglucanase units) and 25 mg ml⁻¹ of proteins. Various tests were done to compare the action of the P. occitanis cellulases with those commercially available and with the traditional stonewashing process. This cellulase preparation was successfully applied in a biostoning process at an industrial scale. The abrasive effect of the P. occitanis cellulases was very uniform and with an efficiency comparable to that obtained by the commercial ones.     

ARTIFICIAL HYBRIDIZATION OF RUBBER-BEARING GUAYULE WITH COLD-TOLERANT PARTHENIUM LIGULATUM

ABSTRACT Caespitose and cold-tolerant plants of Parthenium ligulatum (Jones) Barneby (Asteraceae) from a native population in the Uinta Basin, Utah, were uprooted, potted, and transferred to a greenhouse in California. Approximately two years after transfer, the plants flowered and subsequently were crossed to diploid guayule (Parthenium argentatum Gray), the rubber-bearing species, native to the state of Durango, Mexico. Only female guayule × male P. ligulatum crosses produced F₁ hybrids. Only crosses involving guayule as female parent and F₁ plants as male parents produced backcross (BC,) plants. Hybrid plants were variable with respect to their growth habit, inflorescence, and leaf shape. Both parents and F₁ hybrids had 2n = 36 chromosomes. Unlike the parents, however, meiosis was irregular in the hybrids which showed a range of 0–5 and an average of 2.1 univalents at metaphase I. Hybrids averaged 0.87 laggards at anaphase I and 0.83 micronuclei at the tetrad stage. The crossability of guayule and P. ligulatum, the high degree of chromosome pairing of the F₁ hybrids, and the production of BC₁ plants indicate that the two species are related in spite of their distinct morphological and ecological differences. This study suggests that the cold-tolerance trait of P. ligulatum may be transferred to guayule through interspecific hybridization followed by backcrossing. The development of cold-tolerant guayule cultivars is expected to expand the areas of guayule production beyond that of the Chihuahuan desert and similar climates.     

Feeding by ciliates on two harmful algal bloom species, Prymnesium parvum and Prorocentrum minimum

We have focused on ciliates as potential grazers on toxic phytoplankton because they are major herbivores in aquatic food webs. Ciliates may exert top down control on toxic phytoplankton blooms, potentially suppressing or shortening the duration of harmful algal blooms (HABs). We measured the growth rates of several ciliate species on uni-algal and mixed diets of both HAB and non-HAB algae. The tintinnids Favella ehrenbergii, Eutintinnus pectinis and Metacylis angulata and the non-loricate ciliates Strombidinopsis sp. and Strombidium conicum were isolated from Long Island Sound (LIS), and fed HAB species including the prymnesiophyte Prymnesium parvum (strain 97-20-01) and the dinoflagellate Prorocentrum minimum (strains Exuv and JA 98-01). Ciliates were fed algal prey from cultures at various growth phases and at varying concentrations. We observed no harmful effects of P. minimum (Exuv) on any of the ciliates. However in a comparison of strains, P. minimum (Exuv) supported high growth rates, whereas P. minimum (JA 98-01) supported only nominal growth. P. parvum was acutely toxic to ciliates at high concentrations (2×10⁴–3×10⁴ cells ml⁻¹). At low concentrations (5×10³–1×10⁴ cells ml⁻¹), or in culture filtrate, ciliates survived for at least several hours. In mixed diet experiments, as long as a non-toxic alga was available, ciliates survived and at times grew well at concentrations of P. parvum (5×10²–3×10⁴ cells ml⁻¹) that would otherwise have killed them. The present study suggests that prior to the onset of toxicity and bloom formation ciliates may exert grazing pressure on these HAB species, potentially contributing to the suppression or decline of P. minimum and P. parvum blooms.     

Picolinic acid spray stimulates the antioxidative metabolism and minimizes impairments on photosynthesis on wheat leaves infected by Pyricularia oryzae

Fungal pathogens produce toxins that are important for their pathogenesis and/or aggressiveness towards their hosts. Picolinic acid (PA), a non‐host selective toxin, causes lesions on rice leaves resembling those originated from Pyricularia oryzae infection. Considering that non‐host selective toxins can be useful for plant diseases control, this study investigated whether the foliar spray with PA on wheat (Triticum aestivum L.) plants, in a non‐phytotoxic concentration, could increase their resistance to blast, stimulate the anti‐oxidative metabolism, and minimize alterations in photosynthesis. The PA spray at concentrations greater than 0.1 mg ml^?1 caused foliar lesions, compromised the photosynthesis and was linked with greater accumulation of hydrogen peroxide (H₂O₂) and superoxide anion radical (O₂^??). Fungal mycelial growth, conidia production and germination decreased by PA at 0.3 mg ml^?1. Blast severity was significantly reduced by 59 and 23%, respectively, at 72 and 96 h after inoculation for plants sprayed with PA (0.1 mg ml^?1) at 24 h before fungal inoculation compared to non‐sprayed plants. Reduction on blast symptoms was linked with increases on ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6), glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.8.1.7), glutathione‐S‐transferase (EC 2.5.1.18), peroxidase (EC 1.11.1.7), and superoxide dismutase (EC 1.15.1.1) activities, lower H₂O₂ and O₂^?? accumulation, reduced malondialdehyde production as well as less impairments to the photosynthetic apparatus. A more efficient antioxidative metabolism that rapidly scavenges the reactive oxygen species generated during P. oryzae infection, without dramatically decreasing the photosynthetic performance, was a remarkable effect obtained with PA spray.     

Effects of aflatoxin B1 on in vitro cultures ofNicotiana tabacum var. Samsun

Calli ofNicotiana tabacum (tobacco) were treated with two dose ranges of aflatoxin B₁ (0.1–2.0 µg ml^–1 - low does; 5–25 µg ml^–1 aflatoxin B₁). The ability of calli to recover following 3 weeks of toxin exposure was also investigated. The I₅₀ (50% inhibition) value for fresh mass accumulation was approximately 2 µg ml^–1 AFB₁. Fresh mass accumulation was significantly lower than the control value from 0.5 µg ml^–1 AFB₁. Following 3 weeks growth without a toxin source, the growth of calli up to and including 10 µg ml^–1 AFB₁, was significantly greater than control calli, indicating reversibility of the toxic effects. With increasing toxin concentration, chlorophyll content of callus was inhibited from 0.5 µg ml^–1. Transfer to a toxin-free medium resulted in a degree of recovery (up to 0.5 µg ml^–1). In the dose range 5–25 µg ml^–1, the levels of chlorophyll were drastically reduced, with no recovery following AFB₁ removal. Electron microscopy revealed a disruption of chloroplast structure as an early deteriorative event in AFB₁ exposure of callus cells. Protein levels were less sensitive, with inhibition manifested only in the high dose range. Shoot development occurred at all concentrations, but was significantly inhibited from 5 µg ml^–1 AFB₁. Recovery following toxin removal was minimal at these higher AFB₁ concentrations. The number of necrotic calli increased progressively from 5 µg ml^–1 as toxin levels increased.     

Incidence of charcoal rot of sorghum and soil populations of Macrophomina phaseolina associated with sorghum and native vegetation in Somalia

Studies were conducted in the Bay Region of Somalia to determine the incidence of charcoal rot in sorghum incited by Macrophomina phaseolina and the soil population of M. phaseolina in sorghum fields and areas of native vegetation. Charcoal rot was detected in 34 of 40 sorghum fields. Incidence (percent sorghum hills with diseased plants) in the four regional districts was 21, 70, 20 and 35% (mean of 15 hills/field and 10 fields/district) for Baidoa, Burhakaba, Dinsoore and Quansadhere, respectively. Soil collected from the 40 sorghum fields and from 40 native vegetation sites (10 in each district) all contained M. phaseolina. Mean soil populations for sorghum fields and native vegetation sites were 25.2 and 2.5 microsclerotia g^–1 soil, respectively. Soil populations of M. phaseolina in sorghum fields were significantly (p=0.05) different from populations in native vegetation sites and ranged from 7 to 107 and 0.1 to 14.0 microsclerotia g^–1 soil, respectively. Incidence of charcoal rot in sorghum was positively correlated with soil populations of M. phaseolina (r=0.53, p= 0.01). Correlation between ratoon failure and disease incidence was also highly significant (r=0.48, p=0.01). Correlation between ratoon failure and soil population of M. phaseolina was not significant (r=0.17, p=0.10). Only one out of 30 seed lots of sorghum grown in the Bay Region contained seed infected with M. phaseolina.     

RELATIONSHIP OF WOODY PARTHENIUM ARGENTATUM AND HERBACEOUS P. HISPIDUM VAR. AURICULATUM (ASTERACEAE)

Interspecific hybrids between woody Parthenium argentatum Gray (guayule), native to Mexico and Southwest Texas, and herbaceous perennial P. hispidum var. auriculatum (Britton) Rollins, native to the United States, were obtained successfully. The F₁ hybrids were intermediate for most morphological characters with the exception of the short woody stem, yellow pollen color, and the trichome morphology. Chromosome counts revealed the presence of 2n = 36 A-chromosomes in P. argentatum. The same number of A-chromosomes and four B-chromosomes were found in P. hispidum var. auriculatum. Observations of pollen mother cells showed regular meiosis in both parental species. At diakinesis, chiasmata averaged 1.12 and 1.24 per bivalent for P. argentatum and P. hispidum var. auriculatum, respectively. Meiotic behavior of the F₁ hybrids was irregular. F₁ hybrids averaged 4.43 univalents at metaphase I, 1.95 laggards at anaphase I, and 1.62 micronuclei at the tetrad stage. The low pollen stainability (5.1%) in the F₁ hybrids and the limited number of viable BC₁ seeds (4.07%) may be reflections of the irregular meiosis. Although these primary hybrids are partially fertile, they can be used to introduce desirable characteristics of P. hispidum var. auriculatum, such as herbaceous perennial habit, regrowth ability, and cold tolerance into guayule.     

A selective medium for isolating Rhizoctonia solani from soil

Ko & Hora's (1971) selective medium (FM) for counting Rhizoctonia solani propagules from soil has been modified. FM medium minus gallic acid and fenaminosulf i.e. mineral antibiotic (MA) medium, was amended with inhibitors and fungicides at different concentrations. The modified medium consisting of MA + gallic acid (400 μg ml^-1) + fosetyl-Al (250μgml^-1) was found most efficient in selective isolation of R. solani from soil even in the presence of Macrophomina phaseolina (Rhizoctonia bataticold) and other soil mycoflora. This amended medium was effective in isolating R. solani from soil even when the number of propagules of M. phaseolina present was 10 times greater. The sensitivity of this medium was five times better than Ko & Hora's medium.