Seedling Assay Research Articles

Antagonistic activity of selected strains of Bacillus thuringiensis against Rhizoctonia solani of chili pepper

The aim of this work was to determine, in vitro, the antagonistic effectiveness of 60 strains of Bacillus thuringiensis against damping-off and root and stem rot caused by Rhizoctonia solani. The strains were obtained from the International Collection of Entomopathogenic Bacillus at the FCB-UANL. During the in vitro dual culture assay only 16 of the strains displayed an inhibitory effect. Six strains were chosen to be screened simultaneously by volatile antibiotics, thermostability and seedling assay. In the volatile antibiotics assay, the strains GM-11 and GM-121 showed the best inhibitory effect over R. solani growth. None of the strains showed an efficient antagonistic effect during the thermoestability assay. In seedling assay, majority of the antagonistic isolates, GM-23, GM-11and GM-121, were effective in the reduction of R. solani infection. In addition, GM-23 increased the length of pepper seedlings. These results suggest that the B. thuringiensis strains studied have an excellent potential to be used as bio-control agents of R. solani in chili pepper.

Biological control of Phytophthora blight of pepper by antagonistic rhizobacteria selected from a sequential screening procedure

This study was conducted to select antagonistic rhizobacteria against Phytophthora capsici using a sequential screening procedure and to evaluate control efficacy of drench or root-dip treatments with the selected strains against Phytophthora blight of pepper in the field. Out of 439 bacterial strains, 16 potentially antagonistic strains were screened through radicle and seedling assays and in planta trials, and five candidate strains, CCR04, CCR80, GSE09, ISE13, and ISE14, were selected for field tests. In 2005 and 2006 tests, the control efficacy of the five strains was examined against Phytophthora blight of pepper plants drenched with the bacterial suspension in artificial pathogen inoculation. Three strains, CCR04, CCR80, and ISE14, consistently reduced the disease in both tests. As another form of application, the control efficacy of root-dip treatment was examined on pepper plants just prior to transplanting in the field with natural inoculation in 2006 and 2007. In these tests, four strains, CCR04, CCR80, GSE09, and ISE14, showed consistently good control efficacy against P. capsici, and strains CCR80 and ISE14 increased pepper fruit yield. The strain-treated roots had less infection rates by P. capsici compared with control roots regardless of drench or root-dip treatments. In addition, the strains did not affect the populations of bacteria and fungi in the rhizosphere soil. Therefore, the antagonistic strains selected from the screening procedure provided significant protection against P. capsici through pepper root colonization. These strains could be applied by either drench or root-dip treatment as alternatives to agricultural chemicals to control Phytophthora blight of pepper.

Pathogenicity of Pseudomonas syringae pv. tomato on Tomato Seedlings: Phenotypic and Gene Expression Analyses of the Virulence Function of Coronatine

Bacterial speck disease, which is caused by Pseudomonas syringae pv. tomato, is an economically important disease on tomato. In the present study, we show that P. syringae pv. tomato DC3000 is a pathogen of tomato seedlings, an aspect of pathogen biology that has not been previously investigated. This resulted in the development of a virulence assay on tomato seedlings that has several advantages over labor-intensive foliar assays, including a shorter growth and incubation period, ease of inoculation and handling, and rapid generation of larger sample sizes per experiment. The utility of this assay was investigated by exploring the virulence function of coronatine (COR) on tomato seedlings. Using the COR- mutant DB29 and a MAPMAN display of transcript data from TOM1 microarrays, COR-dependent expression of genes involved in secondary metabolism, polyamine biosynthesis, reactive oxygen species homeostasis, and the novel transcription factor SlNAC2 were identified. Furthermore, during pathogenesis, genes involved in photosynthetic light reactions and the Calvin-Benson cycle were strongly repressed by COR. In conclusion, we show that P. syringae pv. tomato infects tomato seedlings and that COR is required for virulence in seedlings. The seedling assay can be used in high-throughput screens for the identification of molecular targets for COR and for the identification of genes involved in pathogenesis.

Biotypic Diversity in Colorado Russian Wheat Aphid (Hemiptera: Aphididae) Populations

The biotypic diversity of the Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), was assessed in five isolates collected in Colorado. Three isolates, RWA 1, RWA 2, and an isolate from Montezuma County, CO, designated RWA 6, were originally collected from cultivated wheat, Triticum aestivum L., and obtained from established colonies at Colorado State University. The fourth isolate, designated RWA 7, was collected from Canada wildrye, Elymus canadensis L., in Baca County, CO. The fifth isolate, designated RWA 8, was collected from crested wheatgrass, Agropyron cristatum (L.) Gaertn., in Montezuma County, CO. The four isolates were characterized in a standard seedling assay, by using 24 plant differentials, 22 wheat lines and two barley, Hordeum vulgare L., lines. RWA 1 was the least virulent of the isolates, killing only the four susceptible entries. RWA 8 also killed only the four susceptible entries, but it expressed intermediate virulence on seven wheat lines. RWA 6, killing nine entries, and RWA 7, killing 11 entries, both expressed an intermediate level of virulence overall, but differed in their level of virulence to 'CO03797' (Dn1), 'Yumar' (Dn4), and 'CO960293-2'. RWA 2 was the most virulent isolate, killing 14 entries, including Dn4- and Dny-containing wheat. Four wheat lines, '94M370' (Dn7), 'STARS 02RWA2414-11', CO03797, and 'CI2401', were resistant to the five isolates. The results of this screening confirm the presence of five unique Russian wheat aphid biotypes in Colorado.

Comparison of genetic diversity and pathogenicity of fusarium head blight pathogens from China and Europe by SSCP and seedling assays on wheat

Comparison of genetic diversity and pathogenicity of fusarium head blight pathogens from China and Europe by SSCP and seedling assays on wheat

Sequential selection and efficacy of antagonistic rhizobacteria for controlling Phytophthora blight of pepper

This research was conducted to develop a sequential screening procedure for bacteria antagonistic against Phytophthora capsici, and to evaluate control efficacy by candidate antagonistic bacteria selected by this procedure against Phytophthora blight of pepper in the field. A total of 231 bacterial strains were isolated from soils and roots of cucumber, pepper, and tomato plants grown in fields at 12 locations in Korea in 2000 and 2001. From these, 88 strains were pre-selected using a radicle assay. These strains were then screened in a seedling assay with 2-week-old pepper seedlings inoculated with 1000 zoospores of P. capsici g −1 potting mixture, a concentration which caused complete death of the seedlings. Based on the results of this screen, 15 potentially antagonistic strains with at least 50% control efficacy of seedling infection by P. capsici were selected and further tested in 5-week-old pepper plants. In the in planta trials, four bacterial strains, KJ1R5, KJ2C12, KJ9C8, and 11S16, consistently showed significant ( P=0.05) disease suppression against P. capsici infection and were examined as candidate biocontrol agents for their antagonistic effects in the field by artificial inoculation in 2001 and 2002. Plants treated with the bacterial strains KJ1R5, KJ2C12, and KJ9C8 had significantly ( P=0.05) lower disease severity and incidence than untreated controls in at least one test. These field test results have demonstrated that the radicle and seedling assays are very effective as part of the screening procedure to select candidate antagonistic bacteria against P. capsici on pepper. Therefore, effective biocontrol agents for Phytophthora blight of pepper could be selected through the sequential screening procedure developed in this study.

The Iturin and Fengycin Families of Lipopeptides Are Key Factors in Antagonism of Bacillus subtilis Toward Podosphaera fusca

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain. Four Bacillus subtilis strains, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, with proven ability to suppress the disease on melon in detached leaf and seedling assays, were subjected to further analyses to elucidate the mode of action involved in their biocontrol performance. Cell-free supernatants showed antifungal activities very close to those previously reported for vegetative cells. Identification of three lipopeptide antibiotics, surfactin, fengycin, and iturin A or bacillomycin, in butanolic extracts from cell-free culture filtrates of these B. subtilis strains pointed out that antibiosis could be a major factor involved in their biocontrol ability. The strong inhibitory effect of purified lipopeptide fractions corresponding to bacillomycin, fengycin, and iturin A on P. fusca conidia germination, as well as the in situ detection of these lipopeptides in bacterial-treated melon leaves, provided interesting evidence of their putative involvement in the antagonistic activity. Those results were definitively supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of the different lipopeptides. Taken together, our data have allowed us to conclude that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis toward P. fusca.

Rwt4, a wheat gene for resistance to Avena isolates of Magnaporthe oryzae, functions as a gene for resistance to Panicum isolates in Japan

Rwt4 (synonym of Rmg1), a temperature-insensitive gene for resistance to Avena isolates of Magnaporthe oryzae, was identified in wheat cultivar Norin 4 in a seedling assay. The significance of Rwt4 was evaluated using flag leaves of wheat cultivars. At high temperature, Norin 4 was completely resistant to Avena isolate Br58, while Chinese Spring, a noncarrier of Rwt4, was susceptible. Genetic analysis of F2 plants derived from Norin 4 × Chinese Spring indicated that the resistance of flag leaves of Norin 4 to the Avena isolate is conditioned by a single major gene. Segregation analysis of F3 seedlings derived from the F2 plants showed that the major gene is actually Rwt4. These results suggest that Rwt4 is effective against Avena isolates throughout the growth stages. Furthermore, screening of Pyricularia isolates from various hosts suggested that Panicum isolates are possible carriers of the corresponding avirulence gene, PWT4. Segregation analyses of F2 and F3 seedlings showed that Panicum isolates actually carry PWT4, and, therefore, that Rwt4 is also effective against Panicum isolates. On the other hand, none of the Oryza, Setaria, Triticum, and Lolium isolates tested was a carrier of PWT4. The significance of this finding is discussed from the viewpoint of epidemics of blast disease on wheat.

Echinolactones C and D: Two Illudalane Sesquiterpenoids Isolated from the Cultured Mycelia of the Fungus Echinodontium japonicum

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Agrichemical impact on growth and survival of non-target apple phyllosphere microorganisms

The impact of conventional agrichemicals commonly used in New Zealand apple production on non-target, culturable phyllosphere microbial populations was studied in the laboratory (agar, leaf, and seedling assays) and field (apple orchard). Morphologically distinct bacteria (three), yeasts (five), and filamentous microfungi (two) were used as indicator species. The agar assay showed that agrichemical toxicity to microorganisms was dependent on product type, product rate, and organism studied. While the fungicides metiram and captan stopped or severely reduced growth of nearly all microorganisms studied, the insecticides tebufenozide and lufenuron and the fungicide nitrothal-isopropyl showed the least amount of microbial toxicity, each affecting 2 of the 10 indicator organisms studied. In the leaf assay a single agrichemical application at field rate either reduced or increased microbial population counts, again depending on product and microorganism. Repeated agrichemical applications, however, reduced microbial population numbers from 10- to 10,000-fold in planta. Further field research validated these findings, although differences in microbial numbers before and after agrichemical applications were less dramatic. In the orchard, total organism numbers recovered within 2-6 days, but species richness (sum of recognizable taxonomic units) declined. Agrichemicals clearly affected non-target, culturable surface microorganisms. The importance of diversity and stability of microbial populations for disease control still needs to be established.

Differential Selection on Rhynchosporium secalis During Parasitic and Saprophytic Phases in the Barley Scald Disease Cycle

ABSTRACT Competition among eight Rhynchosporium secalis isolates was assessed during parasitic and saprophytic phases of the disease cycle in field experiments conducted at two locations and over two growing seasons. The eight isolates were inoculated onto six barley populations exhibiting varying degrees of resistance. Microsatellite analysis of 2,866 isolates recovered from the field experiments showed significant, and sometimes opposite, changes in the frequencies of R. secalis genotypes during the growing season (parasitic phase) and between growing seasons (saprophytic phase). Isolates that showed the most complex virulence in greenhouse seedling assays had the lowest fitness in the field experiment. Significant differences in isolate fitness were found on different host populations and in different environments. Selection coefficients were large, indicating that evolution can occur rapidly in field populations. Although inoculated isolates had the lowest overall fitness on the moderately resistant landrace cv. Arabi Aswad, some isolates were more virulent and consistently increased in frequency on this landrace, suggesting a risk of directional selection and possible erosion of the resistance following its widespread deployment in monoculture. These results provide the first direct evidence that R. secalis pathogen genotypes differ in their saprophytic ability and parasitic fitness under field conditions.

Echinolactones C and D: Two Illudalane Sesquiterpenoids Isolated from the Cultured Mycelia of the Fungus Echinodontium japonicum

Echinolactones C and D, two compounds with an illudalane sesquiterpenoid skeleton, were isolated from the cultured mycelia of the basidiomycetous fungus Echinodontium japonicum (Echinodontiaceae). The structures of echinolactones C and D were determined by 1D and 2D NMR spectroscopy. Their biological activities were determined using antimicrobial activity and lettuce seedling assays.

Effect of Crop Rotation on Take-all of Wheat in Double-Cropping Systems.

Take-all of wheat (Triticum aestivum), caused by Gaeumannomyces graminis var. tritici, became a serious problem with the widespread adoption of wheat:soybean double-cropping and minimum tillage farming systems in the southeastern United States during the past 30 years. A long-term crop rotation study was initiated in 1994 with 12 double-cropping sequences incorporating wheat, rye, or canola as the fall-planted crop and soybean or grain pearl millet as the summer crop. Cotton and fallow were included in some summer rotations during the last 2 years of the study. The purpose was to identify sustainable alternatives to the continuous wheat:soybean system that would provide acceptable management of take-all. G. graminis var. tritici cultured on autoclaved oats was incorporated into soil prior to planting the first season's crop. Take-all was severe in rotations with continuous wheat each year. Pearl millet was compatible with the cropping system but did not affect incidence or severity of take-all in a following wheat crop. Soybean or pearl millet had little effect on yield loss due to take-all in a subsequent wheat crop. A 1-year rotation with canola significantly reduced take-all incidence and severity. At the end of the second and third seasons, in those rotations where wheat followed 1 year of canola, wheat grain yield was the same as that in control plots that had little or no take-all. Two consecutive years of canola did not suppress take-all or improve wheat yields any more than a single year of canola between wheat crops. Seedling assays for take-all incidence and severity in growth chambers were conducted using soil collected twice each year near the end of each crop's growing season. Results were similar to those observed in the field. However, canola in the rotation had a greater effect in suppressing disease severity than disease incidence. Canola can be a valuable rotational crop for management of take-all in wheat in the southeastern United States.

Relationships among soilborne bean seedling diseases, Lablab purpureus L. and maize stover residue management, bean insect pests, and soil characteristics in Trans Nzoia district, Kenya

Smallholder farmers who practice continuous maize ( Zea mays L.) and bean ( Phaseolus vulgaris L.) cultivation in the highlands of eastern African have been introduced to new leguminous crops for soil fertility enhancement. However, little is known about the impact these crops may have on farmers’ pre-existing crop pest problems. We investigated the cumulative effects of 7 years of differential management of Lablab purpureus L. and maize residues on soilborne bean seedling diseases caused by Pythium spp. and Fusarium solani f. sp. phaseoli. Residue management effects on soil fertility and seedling damage by bean stem maggots ( Ophiomyia spp.) and root-feeding chafer grubs ( Schizonycha spp .) were also evaluated. Differences in disease dynamics were investigated in the laboratory (seed and stem assays). Three consecutive bean seedling crops were grown in the field plots (seedling assays) to assess disease severity and the impact of bean insect pests. The experimental design included three residue management factors: (1) lablab residues removed (L0) or retained (L1); (2) maize stover removed (S0) or retained (S1); and (3) residues left on the soil surface (I0) or incorporated (I1). Residue management practices altered several fundamental soil properties and indirectly affected disease dynamics. Residue incorporation and maize stover removal decreased soil water holding capacity and enhanced Pythium infection of incubated bean seeds. Crop residue removal acidified the soil and reduced nutrient availability, which was linked to a reduction in the number of Fusarium lesions formed on incubated bean stems. In the field, seedling damage by bean fly maggots and chafer grubs increased soilborne disease severity. Lablab residue incorporation plus maize stover removal increased chafer grub abundance. Bean stems incubated in soils from this treatment had significantly more Fusarium lesions, suggesting that differences in relative grub abundance may be related to differential build-up of Fusarium inoculum. Crown and root rot severity of field grown seedlings increased as levels of nutrient availability increased. However, enhanced nutrient availability and maize stover retention reduced disease-induced mortality of the seedlings. Our results point to a complex web of interrelationships among residue management practices, soil characteristics, and soilborne bean seedling pests in Trans Nzoia district Kenya.

Bean seedling damage by root-feeding grubs ( Schizonycha spp.) in Kenya as influenced by planting time, variety, and crop residue management

Use of legume cover crops to improve soil productivity has had unexpected effects on cropping system components in a maize ( Zea mays L.) and bean ( Phaseolus vulgaris L.) cropping system in Trans Nzoia district, Kenya. Smallholder farmers who recently adopted relay cropping of lablab ( Lablab purpureus L.) observed a decline in bean yields where lablab residues had been incorporated prior to maize and bean planting. We investigated chafer grub ( Schizonycha spp.) damage to beans and studied the impact of lablab and maize stover residue management strategy, planting time, and bean cultivar selection on seedling damage. Seedling assays (germination to flower initiation), involving four different bean varieties (GLP2, KK15, KK22, and KTL3), were carried out in three consecutive planting periods. The assays were superimposed on a long-term, factorial experiment where soils had been subjected to differential lablab and maize stover residue management strategies for 7 years. Factors were (1) lablab residues removed (L0) or retained (L1); (2) maize stover removed (S0) or retained (S1) and (3) residues left on the soil surface (I0) or incorporated (I1). Planting time significantly affected the degree of chafer grub damage, with later planted beans sustaining substantially more damage. Of the four bean varieties, KK15 and GLP2 were more grub tolerant than KK22 and KTL3. Grub damage was most severe in plots where lablab residues had been retained and maize stovers had been removed (L1S0). This factor combination had the highest seedling mortality, the poorest stand establishment, and the largest proportion of plants with severed roots systems. The results of this study show that the current farmer practice (lablab residue retention and maize stover removal) is conducive to increased grub-induced bean seedling damage and suggest that early planting, retention of maize stover, and selection of grub-tolerant varieties are important management strategies to reduce damage and subsequent productivity decline.

Fascicularones H—K, Four New Sesquiterpenoids from the Cultured Mycelia of the Fungus Hypholoma fasciculare.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Fascicularones H–K, Four New Sesquiterpenoids from the Cultured Mycelia of the Fungus Hypholoma fasciculare

AbstractThe AcOEt extract of Hypholoma fasciculare yielded four new congeners, fascicularones H (4), I (5), J (6), and K (7). All structures were unambiguously established by 1D and 2D NMR and MS data. In a lettuce seedling assay, compounds 4–7 showed radicle elongation.

Differential response of Amaranthus tuberculatus (Moq ex DC) JD Sauer to glyphosate

Midwest USA farmers have reported inconsistent control of Amaranthus tuberculatus (= rudis) (Moq ex DC) JD Sauer by glyphosate in glyphosate-resistant crops. The potential of selection for decreased A tuberculatus sensitivity to glyphosate was therefore investigated in a reportedly resistant Everly, IA population (P0-EV). Evaluation of six A tuberculatus populations from the Midwest USA estimated a seedling baseline sensitivity of 2.15 mM glyphosate. Based on these results, three generations of divergent recurrent selection were implemented on P0-EV to isolate resistant and susceptible populations. A seedling assay was developed to screen large amounts of seeds and thus expedite the selection process. Whole-plant and seedling rate responses of P0-EV and a known pristine A tuberculatus population from Paint Creek, OH (P0-WT) identified no significant difference in response to glyphosate; however, greater phenotypic variance was ostensibly evident in P0-EV. The first recurrent generation selected for resistance at 3.2 mM glyphosate (RS1-R) had a 5.9- and 1.7-fold resistance increase at the seedling and whole-plant levels, respectively, compared with the susceptible generation selected at 32 microM glyphosate. After three cycles of recurrent selection, 14.6-fold difference in resistance at the seedling level and 3.1-fold difference at the whole-plant level were observed when comparing the populations selected for resistance (RS3-R) and susceptibility (RS3-S). Overall, recurrent selection increased the frequency of resistant individuals and decreased the variability to glyphosate at the population level. Nevertheless, variability for glyphosate resistance was still evident in RS3-R. Results herein suggested that A tuberculatus is inherently variable to glyphosate and that selection decreased the sensitivity to glyphosate. We purport that evolved glyphosate resistance in A tuberculatus may require multiple cycles of selection under field conditions. Historic estimated use of glyphosate alludes to the evolution of tolerant weed populations.

Screening of bacterial antagonists for biological control ofPhytophthora blight of pepper

The aim of this study was to assess the potential of bacterial antagonists to control Phytophthora blight of pepper caused by P. capsici using different screening methods. Among a collection of fluorescent pseudomonas isolated from the rhizosphere of pepper, twelve isolates were initially selected based on dual culture assay on potato dextrose agar and corn meal agar. Further, these twelve isolates were screened for the reduction of disease severity caused by P. capsici using detached leaves and seedling assay. Most of the antagonists showed varying levels of antagonism against P. capsici in both detached leaves and seedlings assay. In addition, few isolates increased shoot and root length of pepper in seedling assays. Among them, isolate PS119 showing highest ability to reduce the disease severity in the in vitro seedling assay was found to be the most efficient antagonists against P. capsici in the in vivo biological control tests. These results indicate that the in vitro seedling assay can be used as a rapid and more accurate technique for the selection of promising biocontrol agents against P. capsici.

Preparation and biological activity of molecular probes to identify and analyze jasmonic acid-binding proteins.

Several types of jasomonic acid (JA) derivatives, including JA--amino acid conjugates, a JA--biotin conjugate, a JA--dexamethasone heterodimer, and a JA-fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA--binding proteins. These JA derivatives, excepting the JA--fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA--binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.