Planta Experiments Research Articles

Evaluation of application methods and biocontrol efficacy of Paenibacillus alvei strain K-165, against the cotton black root rot pathogen Thielaviopsis basicola

Due to limited means for control, the black root rot fungus, Thielaviopsis basicola constitutes a major threat to organic and conventional cotton production worldwide. Despite the substantial yield loss and reduced fiber quality caused by the fungus, little attention has been drawn to the biological control of black root rot disease. In the present study, the efficacy of the biocontrol agent K-165 to control T. basicola on cotton was evaluated. Paenibacillus alvei strain K-165 is a biocontrol agent that has been studied extensively in the past against the soilborne pathogen Verticillium dahliae. K-165 inhibited T. basicola growth in vitro through antibiosis and reduced significantly root discoloration and hypocotyl lesions on cotton seedlings compared to the control treatment, under greenhouse conditions. In the in planta experiments three different application methods of the biocontrol agent were tested. It was shown that a seed coating treatment consisting of K-165 xanthan gum and talc was the most effective in reducing disease symptoms and increasing plant height and fresh weight compared to two other treatments (seed coating or pellets) based on sodium alginate. The efficacy of the K-165 xanthan gum–talc treatment was attributed to the higher bacterial concentration delivered to the seed and the subsequent higher bacterial population on the rhizosphere and soil compared to the other two application methods. K-165 was proven to be an efficient root and soil colonizer inhibiting extensive root colonization by T. basicola as it was shown by qPCR analysis.

Antagonistic Activities of Trichoderma spp. on Phytophthora Root Rot of Sugar Beet

Trichoderma species are among important antagonists of plant pathogenic fungi. The main purpose of this study was to evaluate the biocontrol potential of native Trichoderma spp. isolates from sugar beet fields against Phytophthora drechsleri and P. cryptogea, the causal agents of Phytophthora root rot of sugar beet. During 2007–08, 85 isolates of Trichoderma spp. from sugar beet fields in eight parts of Fars province, Iran were isolated. Based on morphological and physiological characteristics, eight species could be distinguished: T. asperellum, T. atroviride, T. brevicompactum, T. harzianum, T. longibrachiatum, T. spirale, T. tomentosum and T. virens. Their antagonistic activities including competition, colonization and antibiosis against Phytophthora isolates both in vitro and in planta were investigated. Different species and even different isolates of one species differed in their antagonistic capacities. High competitive abilities were shown by T. asperellum, T. atroviride, T. harzianum and T. virens in pre colonized plates, and by the former three species in dual cultures. T. harzianum and T. atroviride efficiently colonized both species of Phytophthora. T. asperellum Ksh2, T. virens DB6r, T. virens DB2 and T. virens DB3 and T. harzianum MS3 gave the highest growth inhibition. In in planta experiments, T. harzianum MS3 and FG4 and T. virens DB6r were the most efficient in suppressing root rot of sugar beet seedlings. Our results suggest that T. harzianum MS3 is the best native candidate for the biocontrol of this disease.

INHIBITION OF OLIVE KNOT DISEASE BY POLYPHENOLS EXTRACTED FROM OLIVE MILL WASTE WATER

Olive mill waste water (OMW) and phenolic extracts, used at different concentrations in vitro displayed a high level of antibacterial activity against Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease. In in planta experiments, phenolic compounds used at three concentrations (1000, 500 and 100 mg l-1) completely inhibited the formation of knots on twigs inoculated with pathogenic strains IVIA 1628 and Aw9. GC/MS analysis revealed the presence of hydroxytyrosol in high concentration, in addition to tyrosol, catechol, caffeic acid and p-coumaric acid, suggesting their antibacterial effect. Copper treatment was less effective than phenolic compounds since the inhibition percentages of knot formation were 6% and 12% for IVIA1628 and Aw9, respectively. These results show that phenolic compounds extracted from OMW and rich in hydroxytyrosol can be excellent substitutes of copper compounds for controlling olive knot disease.

The role of volatile and non-volatile antibiotics produced by Pseudomonas chlororaphis strain PA23 in its root colonization and control of Sclerotinia sclerotiorum

Pseudomonas chlororaphis strain PA23 has demonstrated excellent biocontrol in the canola phyllosphere. This bacterium produces the non-volatile antibiotics phenazine and pyrrolnitrin as well as the volatile antibiotics nonanal, benzothiazole and 2-ethyl-1-hexanol. In vitro experiments were conducted to study the effects of different mutations on the production of these three organic volatile antibiotics by PA23. In planta experiments in the greenhouse investigated the role of the non-volatile antibiotics on root colonization and biocontrol ability of PA23 against Sclerotinia sclerotiorum on sunflower. Analysis of phenazine- and pyrrolnitrin-deficient Tn mutants of PA23 revealed no differences in production of the three volatile antibiotics. On all sampling dates, PA23 applied alone or in combination with the mutants showed significantly higher (P = 0.05) root bacterial number and Sclerotinia wilt suppression (P = 0.05). Decline of the bacterial population seemed to be inversely proportional to/or negatively correlated with the number of antibiotics produced by PA23 but the relative importance of phenazine or pyrrolnitrin on root colonization and/or wilt suppression was not clear. In several cases, the strains producing at least one antibiotic maintained relatively higher bacterial numbers than non-producing strains. However, by 6 weeks after sowing, there was a rapid and significant (P = 0.05) increase in the proportion of introduced bacteria capable of producing at least one antibiotic over the total bacterial population. Furthermore, combining certain mutants with PA23 reduced the root colonization and biocontrol ability of PA23. Strain PA23-314 (gacS mutant) showed competitive colonization in comparison to the other mutants for most sampling dates.

Investigation of horizontal gene transfer potential from Bt176 maize to phytopathogenic bacterium Erwinia stewartii 1082

To analyse the frequency of natural gene transfer from genetically modified maize to phytopathogenic bacterium Erwinia stewartii 1082, a marker rescue system based on the restoration of ampicillin resistance gene was used in in vitro and in planta transformation experiments. A set of three vectors containing defined deletions of the blaTEM116 ampicillin resistance gene in pBR322 was constructed. Recombinant strains of Erw. stewartii 1082 harboring these mutant plasmids were used for infection of transgenic maize plants. Restoration of ampicillin resistance was observed only in transformed electro-competent Erw. stewartii 1082 cells. Frequency of the resistance restoration was found to be dependent on the size of the transforming DNA. In addition, highly active non-specific endodeoxyribonuclease was detected in cell-free lysates of Erw. stewartii 1082, rapidly degrading linear DNA fragments. No ampicillin resistant Erw. stewartii 1082 transformants were observed during in planta experiments indicating that this pathogenic bacterium is not naturally transformable under the conditions tested in this study.

Efficacy of phosphonic acid, metalaxyl‐M and copper hydroxide against Phytophthora ramorum in vitro and in planta

The ability of metalaxyl‐M, phosphonic acid in the form of phosphonate, and copper hydroxide to inhibit different stages in the life cycle of Phytophthora ramorum, the causal agent of sudden oak death (SOD), was tested in vitro using 12 isolates from the North American forest lineage. In addition, experiments were conducted in planta to study the ability of phosphonic acid injections and metalaxyl‐M drenches to control pathogen growth on saplings of California coast live oak (Quercus agrifolia), and of copper hydroxide foliar sprays to control infection of California bay laurel (Umbellularia californica) leaves. Phytophthora ramorum was only moderately sensitive to phosphonic acid in vitro, but was highly sensitive to copper hydroxide. In planta experiments indicated the broad efficacy of phosphonic acid injections and of copper hydroxide sprays in preventing growth of P. ramorum in oaks and bay laurels, respectively. Finally, although metalaxyl‐M was effective in vitro, drenches of potted oak trees using this active ingredient were largely ineffective in reducing the growth rate of the pathogen in planta.

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

Fitness of Isolates of Phytophthora capsici Resistant to Mefenoxam from Squash and Pepper Fields in North Carolina.

Despite the wide adoption of mefenoxam (Ridomil Gold EC) for vegetables in North Carolina, the incidence of Phytophthora blight on pepper (Capsicum annuum) and squash (Cucurbita pepo) is high. Seventy-five isolates of Phytophthora capsici were collected in five pepper and one squash field in order to assess mefenoxam sensitivity. The relative fitness of resistant and sensitive isolates was contrasted in vitro by their respective rates of colony growth and their ability to produce sporangia in unamended V8 juice agar medium. In in vivo experiments, the aggressiveness of isolates on pepper was evaluated. The frequency of resistant isolates in North Carolina populations was 63%, considerably higher than resistance levels in areas where mefenoxam is not widely adopted. Resistant isolates grew on amended media at rates >80 to 90% and >100% of the nonamended control at 100 μg ml-1 and 5 μg ml-1, respectively. Sensitive isolates did not growth at 5 or 100 μg ml-1. All isolates from three fields, including two pepper and a squash field, were resistant to mefenoxam. Populations from other fields were composed of either mixes of sensitive and resistant isolates or only sensitive isolates. Response to mefenoxam remained stable during the course of in vitro and in planta experiments. Occurrence of a mefenoxam-resistant population of P. capsici on squash is reported here for the first time in North Carolina. When measured by rate of colony growth, sporulation in vitro, or aggressiveness in planta, fitness of resistant isolates was not reduced. Mefenoxam-resistant isolates from squash were as aggressive on pepper as sensitive or resistant pepper isolates. These results suggest that mefenoxam-resistant populations of P. capsici are as virulent and fit as sensitive populations.

The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast

In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using freeze-fracture immunolabelling served to detect these proteins in situ. As expected from previous in planta and yeast experiments, NSP accumulated within the nuclei, whereas MP was targetted to the protoplasmic face of plasma membranes when expressed alone. Upon coexpression, NSP was localised at the plasma membranes, where it was strongly attached. These results support a model in which NSP transports viral DNA to the cell periphery to facilitate cell-to-cell movement of viral DNA within plants. In contrast to AbMV MP, no plant-specific protein seems to be necessary for the translocation of NSP to the plasma membrane.

A homologue of the breast cancer-associated gene BARD1 is involved in DNA repair in plants.

hBRCA1 and hBARD1 are tumor suppressor proteins that are involved as heterodimer via ubiquitinylation in many cellular processes, such as DNA repair. Loss of BRCA1 or BARD1 results in early embryonic lethality and chromosomal instability. The Arabidopsis genome carries a BRCA1 homologue, and we were able to identify a BARD1 homologue. AtBRCA1 and the putative AtBARD1 protein are able to interact with each other as indicated by in vitro and in planta experiments. We have identified T-DNA insertion mutants for both genes, which show no visible phenotype under standard growth conditions and are fully fertile. Thus, in contrast to animals, both genes have no indispensable role during development and meiosis in plants. The two single as well as the double mutant are to a similar extent sensitive to mitomycin C, indicating an epistatic interaction in DNA crosslink repair. We could further demonstrate that in Arabidopsis BARD1 plays a prominent role in the regulation of homologous DNA repair in somatic cells.

Characterizing potential bacterial biocontrol agents for suppression of Rhizobium vitis, causal agent of crown gall disease in grapevines

Several bacteria were evaluated in greenhouse studies for their effectiveness at preventing formation of galls induced by Rhizobium vitis, the Gram negative bacterium that causes crown gall disease of grapevines. Three endophytic bacteria isolated from Malus domestica yielded promising results and were selected for further examination: Pseudomonas fluorescens isolate 1100-6, Bacillus subtilis isolate EN63-1, and Bacillus species isolate EN71-1. In planta experiments were initially performed on Nicotiana glauca to facilitate measurement of gall development. The effect of the interval between inoculation of N. glauca with the biocontrol agent and exposure to R. vitis was evaluated. All three bacteria significantly reduced gall size when applied 25 or 86 days before inoculation with R. vitis, and reduced the population of R. vitis relative to control plants . P. fluorescens ‘1100-6’ also reduced the percentage of R. vitis inoculations that yielded galls. P. fluorescens ‘1100-6’ was genetically tagged to allow exploration of its localization in grapevines. In growth chamber studies, P. fluorescens ‘1100-6’ was found to survive in the rhizoplane of grapevines for 6 months. Microscopic examination showed that the genetically modified P. fluorescens ‘1100-6’ predominantly occupied xylem and pith tissues, and occasionally persisted on external surfaces of grapevines.