Effects of transgenic expression of Brevibacterium linens methionine gamma lyase (MGL) on accumulation of Tylenchulus semipenetrans and key aminoacid contents in Carrizo citrange.

KEY MESSAGE: Carrizo transgenic plants overexpressing methionine-gamma-lyase produced dimethyl sulfide. The transgenic plants displayed more resistance to nematode attacks (Tylenculus semipenetrans) and may represent an innovative strategy for nematode control. Tylenchulus semipenetrans is a nematode pest of many citrus varieties that causes extensive damage to commercial crops worldwide. Carrizo citrange vr. (Citrus sinensis L. Usb × Poncirus trifoliate L. Raf) plants overexpressing Brevibacterium linens methionine-gamma-lyase (BlMGL) produced the sulfur volatile compound dimethyl sulfide (DMS). The aim of this work was to determine if transgenic citrus plants expressing BlMGL showed increased tolerance to T. semipenetrans infestation and to determine the effect on the content of key amino acids. While transgenic lines emitted dimethyl sulfide from leaves and roots, no sulfur-containing volatiles were detectable in wild-type Carrizo in the same tissues. Significant changes detected some key amino acids from leaves of transgenic plants such as aspartate, lysine, glycine, leucine and threonine with no changes in the amounts of methionine and α-ketobutyrate. In roots only glycine showed significant changes across all transgenic lines in comparison to wild-type plants. Transgenic plants expressing BlMGL and emitting DMS had less T. semipenetrans aggregation and more biomass than infected WT control plants, indicating that they may represent an innovative management alternative to pesticide/nematicide-based remedies.

Correlations between root-associated microorganisms and peach replant disease symptoms in a California soil.

BACKGROUND: Replant disease often occurs when certain crops are "replanted" in a soil that had previously supported the same or similar plant species. This disease typically leads to reductions in plant growth, crop yields, and production duration, and its etiology remains ill-defined. The objective of this study was to identify microorganisms associated with peach replant disease symptoms at a field location in California, USA. Soil samples were subjected to treatments to create various levels of replant disease symptoms. Clonal peach seedlings were grown in the treated soils in greenhouse trials. After 6 weeks, plant growth parameters were measured, and both culture and culture-independent analyses were performed to identify root-associated bacteria, fungi and stramenopiles. RESULTS: A total of 295,785 bacterial operational taxonomic units (OTU) were identified by an Illumina-based, high throughput sequence analysis of rRNA genes. Among the 60 most abundant OTUs, 27 showed significant (P<0.05) negative correlation with peach shoot weights while 10 were positively correlated. Most of these OTUs belonged to the bacterial phylum Proteobacteria (96%), including the classes Gammaproteobacteria (44.4%), Betaproteobacteria (33.3%) and Alphaproteobacteria (22.2%), and the orders Pseudomonadales, Burkholderiales, Chromatiales, Rhodocyclales, and Sphingomonadales. The most abundant fungi were Trichoderma asperellum, Trichoderma virens, Fusarium oxysporum, Ceratocystis fimbriata and Fusarium solani. The most abundant stramenopiles were Pythium vexans, Pythium violae and an unidentified Aplanochytrium species. Validation experiments using sequence-selective quantitative PCR analyses identified negative and positive associations between P. vexans and Trichoderma spp. and peach shoot weights, respectively. CONCLUSIONS: This study identified numerous microorganisms associated with peach replant symptoms, some of which have been previously identified while others represent new candidates. Subsequent Koch's postulates investigations will assess their possible roles in this replant disease.

Managing nematodes without methyl bromide.

Methyl bromide is an effective pre-plant soil fumigant used to control nematodes in many high-input, high-value crops in the United States, including vegetables, nursery plants, ornamentals, tree fruits, strawberries, and grapes. Because methyl bromide has provided a reliable return on investment for nematode control, many of these commodities have standardized their production practices based on the use of this chemical and will be negatively impacted if effective and economical alternatives are not identified. Alternative control measures based on other chemicals, genetic resistance, and cultural practices require a greater knowledge of nematode biology to achieve satisfactory results. Here, we provide an overview of nematode management practices that we believe will be relied upon heavily in U.S. high-value crop production systems in a world without methyl bromide. Included are case studies of U.S. high-value crop production systems to demonstrate how nematode management practices other than methyl bromide may be incorporated.

Investigations into peach seedling stunting caused by a replant soil.

Replant diseases often occur when pome and stone fruits are grown in soil that had previously been planted with the same or similar plant species. They typically lead to reductions in plant growth, crop yield and production duration. In this project, greenhouse assays were used to identify a peach orchard soil that caused replant disease symptoms. Biocidal treatments of this soil led to growth increases of Nemaguard peach seedlings. In addition, plants grown in as little as 1% of the replant soil exhibited reduced plant growth. These results suggest that the disease etiology has a biological component. Analysis of roots from plants exhibiting various levels of replant disease symptoms showed little difference in the amounts of PCR-amplified bacterial or fungal rRNA genes. However, analysis using a stramenopile-selective PCR assay showed that rRNA genes from this taxon were generally more abundant in plants with the smallest top weights. Nucleotide sequence analysis of these genes identified several phylotypes belonging to Bacillariophyta, Chrysophyceae, Eustigmatophyceae, Labyrinthulida, Oomycetes, Phaeophyceae and Synurophyceae. Sequence-selective quantitative PCR assays targeting four of the most abundant phylotypes showed that both diatoms (Sellaphora spp.) and an oomycete (Pythium ultimum) were negatively associated with plant top weights.

Virulence of Meloidogyne spp. and Induced Resistance in Grape Rootstocks.

Harmony grape rootstock displays resistance to several Meloidogyne spp. but that resistance is not durable in commercial vineyard settings. A 2-year experiment in a microplot setting revealed host specificities of two virulent populations of Meloidogyne arenaria and an avirulent population of Meloidogyne incognita. In a subsequent split-root experiment, the avirulent nematode population was demonstrated to induce resistance to the virulent nematode population. To quantify the level of resistance, reproduction of the virulent nematode population was determined 63 days after being challenged by an avirulent nematode population using a range of inoculum densities and timeframes. Induction of resistance became apparent when the virulent nematode population was inoculated 7 days after the avirulent nematode population and increased thereafter. The level of induced resistance increased with increased inoculum levels of the avirulent nematode population. Root systems of perennial crops are commonly fed upon simultaneously by multiple nematode species. These two studies indicate that field populations can become preferentially virulent upon one or multiple rootstocks and that co-inhabiting populations may induce existing resistance mechanisms. In perennial crops, it is common for numerous nematode species besides Meloidogyne spp. to be present, including some that feed without causing apparent damage.

Influence of Ring Nematode Infestation and Calcium, Nitrogen, and Indoleacetic Acid Applications on Peach Susceptibility to Pseudomonas syringae pv. syringae.

ABSTRACT Two field experiments were conducted to study the effects of added nitrogen, calcium, and indoleacetic acid, in the presence or absence of ring nematodes (Mesocriconema xenoplax), on susceptibility of peach to bacterial canker. When noninfested soil was inoculated with ring nematodes, peach tree susceptibility to bacterial canker infection caused by Pseudomonas syringae pv. syringae was dramatically increased after a period of 2 years. However, no evidence was found that ring nematode infestation increased tree water stress or, in turn, altered plant calcium uptake. Soil fumigation with methyl bromide prior to planting in a commercial orchard significantly reduced both nematode populations and peach tree susceptibility to bacterial canker infection when compared with nonfumigated treatments. In both experiments, tree susceptibility, as measured by canker length following inoculation of stems with P. syringae pv. syringae, was negatively correlated with plant tissue nitrogen content and positively correlated with tissue calcium content. A principal components analysis showed that tissue nitrogen and calcium levels were negatively correlated, and that high-nitrogen, low-calcium tissues were less susceptible to bacterial canker than low-nitrogen, high-calcium tissues. These results indicate that the increased susceptibility of peach to P. syringae pv. syringae under nematode infestation conditions is mediated by both nutritional effects (primarily nitrogen) and nutritional-independent effects, but do not support previous reports of beneficial effects of calcium for reducing bacterial canker.

Interaction Between Nitrogen-Fertilized Peach Trees and Expression of syrB, a Gene Involved in Syringomycin Production in Pseudomonas syringae pv. syringae.

ABSTRACT The in vitro expression of the syrB gene that controls the synthesis of syringomycin, a non-host-specific phytotoxin produced by Pseudomonas syringae pv. syringae van Hall, was studied using aqueous extracts derived from bark tissues collected from nitrogen-fertilized and nonfertilized peach trees. Expression of the syrB gene was quantified as beta- galactosidase activity expressed by P. syringae pv. syringae B3AR-132 containing a syrB::lacZ fusion. Gene expression was significantly less in three of four paired comparisons using extracts derived from fertilized versus nonfertilized trees; however, canker lengths were significantly different in only one of four comparisons. Expression was negatively correlated with plant tissue nitrogen content and positively correlated with a plant carbon/nitrogen ratio. Bark tissue from ring nematodeinfested trees had significantly higher concentrations of total soluble phenolic compounds and carbon/nitrogen ratios than bark samples from trees without nematodes, and canker size was significantly greater in trees growing in ring nematode-infested soil compared with noninfested soil. Nitrogen fertilization significantly decreased the plant carbon/nitrogen ratio, which was positively correlated with the concentration of total soluble phenolic compounds. Canker size developing after bacterial inoculation was positively correlated with higher plant carbon/nitrogen ratios and total soluble phenolic compounds. These results support the hypothesis that one reason why nitrogen fertilization decreases host susceptibility to bacterial canker is by either reducing the amount of plant metabolites that can induce syrB gene expression, or producing or increasing the concentration of compounds that antagonize syrB inducing compounds.