Cruciferous Weeds Do Not Act as Major Reservoirs of Inoculum for Black Rot Outbreaks in New York State.

Cruciferous weeds have been shown to harbor diverse Xanthomonas campestris pathovars, including the agronomically damaging black rot of cabbage pathogen, X. campestris pv. campestris. However, the importance of weeds as inoculum sources for X. campestris pv. campestris outbreaks in New York remains unknown. To determine if cruciferous weeds act as primary reservoirs for X. campestris pv. campestris, fields that were rotating between cabbage or had severe black rot outbreaks were chosen for evaluation. Over a consecutive 3-year period, 148 cruciferous and noncruciferous weed samples were collected at 34 unique sites located across five New York counties. Of the 148 weed samples analyzed, 48 X. campestris isolates were identified, with a subset characterized using multilocus sequence analysis. All X. campestris isolates originated from weeds belonging to the Brassicaceae family, with predominant weed hosts being shepherd's purse (Capsella bursa-pastoris), wild mustard (Sinapis arvensis), yellow rocket (Barbarea vulgaris), and pennycress (Thlaspi arvense). Identifying pathogenic X. campestris weed isolates was rare, with only eight isolates causing brown necrotic leaf spots or typical V-shaped lesions on cabbage. There was no evidence of cabbage-infecting weed isolates persisting in an infected field by overwintering in weed hosts; however, similar cabbage and weed X. campestris haplotypes were identified in the same field during an active black rot outbreak. X. campestris weed isolates are genetically diverse both within and between fields, but our findings indicate that X. campestris weed isolates do not appear to act as primary sources of inoculum for B. oleracea fields in New York.

Introgression and QTL mapping conferring resistance for Alternaria brassicae in the backcross progeny of Sinapis alba + Brassica juncea somatic hybrids.

KEY MESSAGE: A total of three QTLs, responsible for A. brassicae resistance were introgressed into S. alba - B. juncea introgression lines from S. alba and mapped through donor genome-specific SSR markers. Alternaria brassicae is a key pathogen of the Brassicaceae family causing severe blight disease to oilseed crops that leads to heavy yield losses due to lack of resistance source within cultivated Brassicas. However, the host resistance present in the Sinapis alba, an allied member of the Brassicaceae family is still unexplored precisely due to the unavailability of segregating population for Alternaria blight resistance and scarcity of donor genome-specific genetic markers. The present study was undertaken to identify quantitative trait loci governing resistance to Alternaria blight which was introgressed from S. alba to the backcross population of stable S. alba + B. juncea somatic hybrids (2n = 60; AABBSS). The second backcross population showed significant phenotypic variations for Alternaria blight ranging from immune to highly susceptible phenotype, thus suggesting quantitative nature of resistance for the disease. A subset of 154 BC2F3-4 lines was used for disease screening and genotyping with 234 S. alba genome-specific microsatellite markers. As a result of the study, twelve linkage groups were developed corresponding to 12 chromosomes of S. alba (n = 12) covering a length of 1694.02 cM. The chromosomes 5 and 11 harbored a total of 1 (Abr-01), and 2 (Abr-02, and Abr-03) QTLs detected by ICIM-ADD mapping method at LOD score values 3.7, 5.12, and 6.74, respectively. The QTLs identified during the study have a range of 5.51-10.87 percent phenotypic variations for disease resistance. To the best of our knowledge, this is the first report of QTLs introgression for A. brassicae resistance in cultivated Brassica from an allied member of Brassicaceae.

Evaluation of physiological and molecular effect of variable virulence of Alternaria brassicae isolates in Brassica juncea, Sinapis alba and Camelina sativa.

Brassica genus comprises many prominent species valuable for human nutrition including vegetable crops and oilseed. Production of B. juncea is challenged by many abiotic and biotic stresses, Alternaria blight caused by a necrotrophic fungal pathogen Alternaria brassicae is one of the most prominent diseases of cruciferous crops including B. juncea. However, some closely related wild species like Sinapis alba and Camelina sativa exhibit a variable level of resistance towards the pathogen. Apart from the host resistance, intra-specific pathogen variability also influences disease severity to a larger extent. In this study, we identified and isolated two strains of A. brassicae viz ABS1 and ABS2 exhibiting morphological and pathological variability. These isolates were further used to artificially inoculate B. juncea and two of its wild relatives under in-vitro as well as in-vivo conditions to inspect their pathogenicity in a susceptible, a moderately resistant and a highly resistant host. virulent isolate (ABS2) was able to readily establish infection in all the three species whereas the less virulent isolate (ABS1) readily infected susceptible species B. juncea but delayed and mild infection was noticed in tolerant hosts. Variable physiological and molecular host response towards the differential level of virulence of pathogen were established with many confirmatory experiments like DAB staining study, Disease severity index and microscopic analysis. Real-time PCR results confirm that these two isolates induce a variable level of induction in genes PR1 and PDF1.2 within 48 h of the artificial inoculation in B. juncea and its wild relatives.

A combined transcriptional, biochemical and histopathological study unravels the complexity of Alternaria resistance and susceptibility in Brassica coenospecies.

Alternaria blight is one of the most devastating diseases of rapeseed-mustard caused by a necrotrophic fungus Alternaria brassicae. Lack of satisfactory resistance resource in Brassica is still a main obstruction for developing resistance against Alternaria. In this study, we have selected Brassica juncea, Sinapis alba and Camelina sativa to understand and unravel the mechanism of disease resistance against Alternaria. Histopathological studies showed early onset of necrosis in B. juncea (1 dpi) and delayed in S. alba (2 dpi) and C. sativa (3 dpi) respectively. Early and enhanced production of hydrogen peroxide (H2O2) was observed in C. sativa and S. alba (6 hpi) when compared to B. juncea (12 hpi). An increase in catalase activity was observed in both C. sativa (36 % at 6 hpi) and S. alba (15 % at 12 hpi), whereas it significantly decreased in B. juncea at 6 hpi (23 %), 12 hpi (30 %) and 24 hpi (8 %). Gene expression analysis showed induction of PR-3 and PR-12 genes only in C. sativa and S. alba when compared to B. juncea suggesting their vital role for Alternaria resistance. In contrast, SA marker genes were significantly expressed in B. juncea only which provides evidence of hormonal cross talk in B. juncea during Alternaria infection thereby increasing its susceptibility.

Diversity of organotrophic bacteria, activity of dehydrogenases and urease as well as seed germination and root growth Lepidium sativum, Sorghum saccharatum and Sinapis alba under the influence of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are organic compounds with highly toxic, carcinogenic, and mutagenic properties, which adversely affect the basic biological parameters of the soil, including the count of microorganisms, and the enzymatic activity. In addition to disturbances to the biological activity of the soil, PAHs may also exhibit toxic effects on plants. In view of the above, the study involved testing aimed at the determination of the effects of polycyclic aromatic hydrocarbons in a form of naphthalene, phenanthrene, anthracene and pyrene on the count, colony development (CD) index, ecophysiological (EP) diversity index of organotrophic bacteria, and the activity of soil dehydrogenases and soil urease. Moreover, an attempt was made to determine the soil's resistance based on the activity of the above-listed enzymes, and the effect of polycyclic aromatic hydrocarbons on seed germination and root growth was assessed by Lepidium sativum, Sorghum saccharatum, and Sinapis alba. In addition, the species of bacteria found in a soil subjected to strong pressure of polycyclic aromatic hydrocarbons were isolated. The experiment was performed in a laboratory on samples of loamy sand. Polycyclic aromatic hydrocarbons were introduced into the soil in an amount of 0, 1000, 2000, and 4000 mg kg(-1) of soil dry matter. Germination and growth of cress (L. sativum), white mustard (S. alba), and sweet sorghum (S. saccharatum) were determined using Phytotoxkit tests. It was found that the tested PAHs increased the average colony counts of organotrophic soil bacteria; pyrene did so to the greatest extent (2.2-fold relative to non-contaminated soil), phenanthrene to the smallest extent (1.4-fold relative to non-contaminated soil). None of the PAHs changed the value of the bacterial colony development (CD) index, while anthracene and pyrene increased the value of the eco-physiological (EP) diversity indicator. PAHs lowered the activity of the tested enzymes. The activity of dehydrogenases was dependent on a greater extent by the type of hydrocarbon (54.56%) rather than by the dose (10.64%), while for the activity of urease, it was the opposite. The greater extent was dependent on dose (95.42%) rather than by type (0.21%). Dehydrogenases are characterised by greater resistance to the action of PAHs than urease. Based on seed germination and root growth, it has shown that S. alba is best suited, being the most vulnerable plant, while S. saccharatum is the least suited. Subjecting a soil to strong pressure of PAHs leads to disturbances to the biological parameters of the soil, seed germination, and root growth L. sativum, S. saccharatum, and S. alba.

Salicylic acid-mediated establishment of the compatibility between Alternaria brassicicola and Brassica juncea is mitigated by abscisic acid in Sinapis alba.

This work addresses the changes in the phytohormonal signature in the recognition of the necrotrophic fungal pathogen Alternaria brassicicola by susceptible Brassica juncea and resistant Sinapis alba. Although B. juncea, S. alba and Arabidopsis all belong to the same family, Brassicaceae, the phytohormonal response of susceptible B. juncea towards this pathogen is unique because the latter two species express non-host resistance. The differential expression of the PR1 gene and the increased level of salicylic acid (SA) indicated that an SA-mediated biotrophic mode of defence response was triggered in B. juncea upon challenge with the pathogen. Compared to B. juncea, resistant S. alba initiated enhanced abscisic acid (ABA) and jasmonic acid (JA) responses following challenge with this pathogen, as revealed by monitoring the expression of ABA-related genes along with the concentration of ABA and JA. Furthermore, these results were verified by the exogenous application of ABA on B. juncea leaves prior to challenge with A. brassicicola, which resulted in a delayed disease progression, followed by the inhibition of the pathogen-mediated increase in SA response and enhanced JA levels. Therefore, it seems that A. brassicicola is steering the defence response towards a biotrophic mode by mounting an SA response in susceptible B. juncea, whereas the enhanced ABA response of S. alba not only counteracts the SA response but also restores the necrotrophic mode of resistance by enhancing JA biosynthesis.

Preservation of pears in water in the presence of Sinapis arvensis seeds: a Greek tradition.

In this research, the microbiological and physicochemical changes during preservation of pears in water in the presence of Sinapis arvensis seeds (PWS FL) according to the traditional Greek home food manufacture were studied. Pears preserved in water served as control (PW FL). The growth of lactic acid bacteria (LAB) coming from the pear surface was enhanced in the presence of Sinapis seeds, while Enterobacteriaceae and Gram-negative bacteria declined coincidently with the lower (P<0.05) pH of the PWS FL. LAB predominated over the other microbial groups in the fermentation liquids (FLs) of both systems. All the 49 LAB isolates from one fermentation experiment were identified as Leuconostoc mesenteroides subsp. cremoris by the SDS-PAGE of whole-cell proteins, while RAPD-PCR fingerprinting and partial 16S rRNA sequence determination of selected isolates did not discriminate them at the subspecies level. Fruit preserved in PWS FL had higher titratable or volatile acidity, phenolic compounds or antioxidant capacity as well as lower pH and firmness than the control fruit. All physicochemical parameters of the FLs increased except of the pH which decreased. Coincidently with higher population of LAB in PWS FL the levels of citric, lactic and acetic acid were higher than in control. Oxalic acid and related unknown substances were found at higher levels in PWS FL than the control and may be the agent(s) enhancing the growth of LAB and/or contributing partially to the decline of Enterobacteriaceae. The organoleptic test showed that fruit preserved in PWS FL had better overall acceptance than the control, and that it retained most of the positive traits.

Differential profiling of selected defence-related genes induced on challenge with Alternaria brassicicola in resistant white mustard and their comparative expression pattern in susceptible India mustard.

The lack of availability of sources of resistance against Alternaria brassicicola within the family Brassicaceae has made oilseed mustard plants a target for one of the most damaging and widespread fungal diseases, Alternaria black spot. Of the other non-host-resistant/tolerant plants, Sinapis alba, white mustard, is considered to be the most important apart from Arabidopsis. To understand the defence response of S. alba upon incompatible interaction with this pathogen, a functional genomic approach using cDNA amplified fragment length polymorphism was performed. The highly reproducible bands, found to be either more amplified or uniquely present in infected S. alba plants compared with non-infected plants, were further subjected to comparative reverse Northern analysis in the incompatible white mustard (S. alba) and compatible India mustard (Brassica juncea L.) plants. The suppression of 46% of the genes in the compatible background indicates the possibility of effective and specific recognition of Alternaria in S. alba. Analysis of the 118 genes up-regulated specifically in infected S. alba compared with B. juncea showed that 98 genes have similarity to proteins such as receptor-like protein kinase genes, genes involved with calcium-mediated signalling and salicylic acid-dependent genes as well as other genes of known function in Arabidopsis. The apparent expression profile data were further confirmed for selected genes by quantitative real-time polymerase chain reaction analysis. Classification of these genes on the basis of their induction pattern in Arabidopsis indicates that the expression profile of several of these genes was distinct in S. alba compared with B. juncea.

Molecular interaction between Methylobacterium extorquens and seedlings: growth promotion, methanol consumption, and localization of the methanol emission site.

Four Methylobacterium extorquens strains were isolated from strawberry (Fragaria x ananassa cv. Elsanta) leaves, and one strain, called ME4, was tested for its ability to promote the growth of various plant seedlings. Seedling weight and shoot length of Nicotiana tabacum, Lycopersicon esculentum, Sinapis alba, and Fragaria vesca increased significantly in the presence of the pink-pigmented facultative methylotroph (PPFM), but the germination behaviour of seeds from six other plants was not affected. The cell-free supernatant of the bacterial culture stimulated germination, suggesting the production of a growth-promoting agent by the methylotroph. Methanol emitted from N. tabacum seedlings, as determined by proton-transfer-reaction mass spectrometry (PTR-MS), ranged from 0.4 to 0.7 ppbv (parts per billion by volume), while significantly lower levels (0.005 to 0.01 ppbv) of the volatile alcohol were measured when the seedlings were co-cultivated with M. extorquens ME4, demonstrating the consumption of the gaseous methanol by the bacteria. Additionally, by using cells of the methylotrophic yeast Pichia pastoris transformed with the pPICHS/GFP vector harbouring a methanol-sensitive promoter in combination with the green fluorescence protein (GFP) reporter gene, stomata were identified as the main source of the methanol emission on tobacco cotyledons. Methylobacterium extorquens strains can nourish themselves using the methanol released by the stomata and release an agent promoting the growth of the seedlings of some crop plants.

Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity.

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.

Plant-assisted degradation of phenanthrene as assessed by solid-phase microextraction (SPME).

The soil bacterium Sphingomonas yanoikuyae was isolated from a petroleum-contaminated soil and grown on mineral salts agar overlaid with the polycyclic aromatic hydrocarbon phenanthrene. The effect of white mustard, Sinapis alba, on phenanthrene degradation by S. yanoikuyae in artificially contaminated Redi-earth-sand was examined. Solid-phase-microextraction (SPME) gas chromatography-flame ionization detection (GC-FID) was used to quantify the concentration of phenanthrene in the gas phase of Magenta jars containing S. alba and S. yanoikuyae, each alone and with no additions. Gas chromatography-mass spectrometry (GC-MS) of Soxhlet extracts was used to determine the concentration of phenanthrene remaining in Redi-earth-sand. The gas phase concentration of phenanthrene in nonsterile Redi-earth-sand decreased by 99.7% in treatments with S. alba plus S. yanoikuyae, by 98.6% with S. alba, by 96.7% with S. yanoikuyae, and by 95.8% with no additions. Under gnotobiotic conditions, the gas phase concentration of phenanthrene in Redi-earth-sand decreased by 94% in treatments with S. alba plus S. yanoikuyae, by 77% with S. yanoikuyae, by 26% with S. alba, and 0% with no additions. The concentration of phenanthrene in Redi-earth-sand under gnotobiotic conditions decreased in treatments with S. alba plus S. yanoikuyae by 88%, by 67% with S. yanoikuyae, by 13% with S. alba, and 0% with no additions as measured in Soxhlet extracts. These results suggest that SPME-GC can be used to rapidly assess the potential of plants and microorganisms to reduce the level of unaged polyaromatic hydrocarbons such as phenanthrene in soil. This method provided results that were consistent with the more costly Soxhlet extraction method and was less time consuming.