Chitin elicitor receptor kinase 1 (CERK1) is required for the non-host defense response of <Emphasis Type="Italic">Arabidopsis</Emphasis> to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">cubense</Emphasis>

Banana wilt disease is a typical vascular disease caused by the fungal pathogen Fusarium oxysporum f. sp. cubense 4 (Foc 4). Pattern recognition receptors in the plant cell membrane can recognize pathogen-associated molecular patterns (PAMPs) to activate multi-layer defense responses, including defense gene expression, stomatal closure, reactive oxygen species (ROS) burst and callose deposition, to limit pathogen growth. In the present study, we found that chitin elicitor receptor kinase 1 (CERK1) was required for the non-host resistance of Arabidopsis thaliana to Foc B2 (a strain of Foc 4). The cerk1 mutant had weaker defense responses after Foc B2 treatment, including lower expression of PAMP- and salicylic acid-responsive genes, no stomatal closure, lower ROS level and less callose deposition, than that of the wild-type plant. Consistent with this, the cerk1 mutant plants exhibited higher susceptibility to non-host pathogen Foc B2. These results suggest the crucial importance of CERK1 in Foc B2-triggered non-host resistance.     

Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

Cytological karyotyping of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> by the germ tube burst method (GTBM)

Fusarium oxysporum is an ascomycete fungus including plant pathogenic and nonpathogenic strains. Genome analyses have indicated that the karyotype of F. oxysporum is diverse among isolates. Here we used the germ tube burst method (GTBM), a more reliable method than conventional cytology or pulsed field gel electrophoretis, to karyotype isolates of F. oxysporum ff. spp. lycopersici and conglutinans and nonpathogenic F. oxysporum. In this first application of GTBM for F. oxysporum, pathogenic isolates were found to have more chromosomes than in nonpathogenic isolates. We also used a ribosomal DNA probe and fluorescence in situ hybridization (FISH) to analyze chromosome structure.     

<Emphasis Type="Italic">Fusarium ershadii</Emphasis> sp. nov., a Pathogen on <Emphasis Type="Italic">Asparagus officinalis</Emphasis> and <Emphasis Type="Italic">Musa acuminata</Emphasis>

Two Fusarium strains, isolated from Asparagus in Italy and Musa in Vietnam respectively, proved to be members of an undescribed clade within the Fusarium solani species complex based on phylogenetic species recognition on ITS, partial RPB2 and EF-1α gene fragments. Macro- and micro-morphological investigations followed with physiological studies done on this new species: Fusarium ershadii sp. nov can be distinguished by its conidial morphology. Both isolates of Fusarium ershadii were shown to be pathogenic to the monocot Asparagus officinalis when inoculated on roots and induced hollow root symptoms within two weeks in Asparagus officinalis seedlings. In comparison mild disease symptoms were observed by the same strains on Musa acuminata seedlings.     

Identification of <Emphasis Type="Italic">Fusarium</Emphasis> species associated with soybean root rot in Sichuan Province,China

Soybean root rot is a worldwide soil-borne fungal disease threatening soybean production, causing large loss in yield and quality of soybean. Fusarium species are well recognized as the important causal agent of Fusarium root rot, which are often distinct with respect to various factors in different soybean-producing regions around the world. Recently, Fusarium root rot has been frequently reported in Sichuan Province of China, where is unique in its climate and diverse cropping patterns, but it is still unclear about the predominant Fusarium species and their pathogenicity on soybean. In this study, diseased soybean roots were collected from three regions of Sichuan Province during 2014–2015. Based on morphological characteristics and phylogenetic analysis of nucleotide sequences of the ribosomal internal transcribed spacer region and the translation elongation factor 1-α gene, 78 isolates of Fusarium were identified as nine distinct species. Pathogenicity tests showed that seven species of Fusarium were able to infect soybean, but differed in pathogenicity. F. oxysporum, F. equiseti and F. graminearum were the most aggressive species to soybean, whereas F. fujikuroi and F. verticillioides were not pathogenic to soybean. There was a strong positive correlation of the pathogenicity of Fusarium species with seedling emergence and fresh root weight. In addition, the diversity of Fusarium species varied among soybean-growing regions. To our knowledge, this report on population and pathogenicity of Fusarium species, in particular, F. graminearum, associated with soybean root rot in Sichuan Province of southwest China, will be helpful to provide effective control strategies for the disease.     

A recombinant flagellin fragment,which includes the epitopes flg22 and flgII-28, provides a useful tool to study flagellin-triggered immunity

Plants and animals independently evolved the ability to recognize flagellin (also called FliC), the building block of the bacterial flagellum, as part of their innate immune response. While animals recognize a relatively large region of FliC, most plants recognize one or two short epitopes of FliC: flg22 and flgII-28. However, since most research in plants has focused on flg22 and flgII-28 and not the actual FliC protein, the importance of any FliC region beyond the two epitopes in plant immunity is poorly understood. Here we report cloning, overexpression, and purification of a Pseudomonas syringae FliC fragment from amino acid 1 to 143, which includes both FliC epitopes and the adjacent alpha helices. Exposing Arabidopsis thaliana leaves to FliC_1–143 did not reveal any additional FliC recognition capabilities beyond flg22. However, while the kiwifruit species Actinidia arguta did not respond to either flg22 or flgII-28, treatment of A. arguta leaves with FliC_1–143 triggered a significant reactive oxygen response, indicating recognition. This result suggests that in some plant species, recognition of FliC requires regions of FliC beyond the two well-known epitopes and that FliC_1–143 represents a useful tool in the study of plant immunity.     

Root-specific expression of defensin in transgenic tobacco results in enhanced resistance against <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Phytophthora species are soil-borne pathogens that damage plants in both agro- and natural ecosystems. To suppress the devastating pathogen, we generated a root-specific expression system using a specific promoter (pPRP3) conferring elevated expression of the target gene in roots that are very susceptible to soil-borne pathogens. To verify root-specific expression, we compared β-glucuronidase (GUS) expression driven by a constitutive or root-specific promoters in shoots and roots. In histochemical and fluorometric assays, GUS activity was detected in whole tobacco plants when GUS expression was driven by p35S, but was detected only in the roots by pPRP3. We then expressed a pepper defensin (J1–1) gene in tobacco to elucidate its effect on plant resistance. The accumulation of J1–1 was also tissue-specific in transgenic tobacco plants. Finally, transgenic plants carrying GUS or J1–1 genes in combination with p35S or pPRP3 were inoculated with Phytophthora parasitica var. nicotianae and Pythium aphanidermatum. Disease symptoms were significantly suppressed in transgenic plants that accumulated J1–1, regardless of the promoter used. Furthermore, the expression of PR genes was induced in J1–1 transgenic plants, exhibiting much higher levels in p35S-driven J1–1 plants than in pPRP3::J1–1 plants. These results demonstrated that J1–1 transgenic plants were primed for enhanced expression of PR genes, which provided synergistic effects with the defensin for disease resistance.     

Diversity of <Emphasis Type="Italic">Fusarium</Emphasis> species associated with root rot of sugar beet in China

Sugar beet is widely grown throughout the world and represents the second largest crop used to produce sugar. Root rot in sugar beet, caused by Fusarium, significantly reduces yield, juice purity, and sugar concentration. Here, 307 Fusarium isolates were collected from sugar beet roots exhibiting typical root rot symptoms in eight provinces or autonomous regions of China from 2009 to 2012. Based on morphological characteristics and sequence data of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) and the translation elongation factor 1α (EF-1α), Fusarium oxysporum (38.4%) was identified as the most prevalent species, followed by F. solani (20.9%), and F. equiseti (18.9%). These three species were widely distributed in all eight of the provinces and autonomous regions. F. tricinctum (5.9%), F. brachygibbosum (4.6%), F. redolens (3.3%), F. proliferatum (3.3%), F. graminearum (2.3%), F. verticillioides (1.6%), F. nygamai (0.7%), and F. culmorum (0.3%) were less frequently obtained. Of the 307 Fusarium isolates, 117 representing different species and geographic locations were demonstrated to cause tip rot and vascular discoloration in sugar beet roots, with disease incidence ranging from 84.2 to 100.0% and disease index ranging from 41.94 to 75.83. This is the first detailed report of Fusarium species, in particular F. tricinctum, F. brachygibbosum, F. redolens, F. proliferatum, F. nygamai, and F. culmorum, causing sugar beet root rot in China.     

<Emphasis Type="Italic">Bradyrhizobium</Emphasis> isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.     

Occurrence and characterisation of <Emphasis Type="Italic">Rhizoctonia</Emphasis> species causing diseases of ornamental plants in Italy

During surveys conducted in 2010–2012 Rhizoctonia symptoms were observed on 30 ornamental species in different nurseries located in eastern Sicily (Southern Italy). Eighty-eight isolates of Rhizoctonia spp. were obtained from symptomatic leaves, roots and stems. Fifty-six of the isolates were binucleate and 32 were multinucleate Rhizoctonia. Characterisation of anastomosis groups (AGs) was performed using morphological characteristics and sequence analysis of the internal transcribed spacer of ribosomal DNA ( rDNA-ITS) region. Most isolates collected were Rhizoctonia solani AG-4 HG-I (35.2% of all isolates) and one isolate was AG-2-2 IIIB. The binucleate isolates belonged to AG-R (27.3%), AG-A (21.6%), AG-G (12.5%), AG-V (1.1%) and AG-Fb (1.1%). The pathogenicity of 38 representative isolates collected from each host was tested on seedlings or cuttings grown in a growth chamber. All R. solani AG-4 HG-I isolates, most of the binucleate AG-R, AG-A and AG-G and AG-V were pathogenic and reproduced symptoms identical to that observed in nurseries, while binucleate AG-Fb and R. solani AG-2-2 IIIB isolates were nonpathogenic. This is the first report of the occurrence of Rhizoctonia species on some ornamental plants and the first report of binucleate Rhizoctonia AG-R and AG-V in Europe.     

Seasonal progression of leaf rust in ‘Niagara Rosada’ grapevine in a biannual crop system in Brazil

The soil-borne fungus Fusarium oxysporum can cause both Fusarium yellows and Fusarium root rot diseases with severe yield losses in cultivated sugar beet. These two diseases cause similar foliar symptoms but different root response and have been proposed to be caused by two distinct F. oxysporum formae speciales. Fusarium yellows, caused by F. oxysporum f. sp. betae, presents vascular discoloration, whereas Fusarium root rot, due to F. oxysporum f. sp. radicis-betae, appears as black rot visible on the root surface. The aim of this work was to study the host-pathogen interaction between sugar beet lines and isolates originally characterized as Fusarium oxysporum f. sp. betae. Eight susceptible sugar beet lines, selected by the USDA-ARS (US) and UNIPD (University of Padova, Italy) breeding programs, were inoculated with three different isolates of F. oxysporum f. sp. betae, the causal agent of Fusarium yellows, representing different genetic groups. All inoculated lines developed symptoms, but severity, expressed as area under the disease progress curve (AUDPC), differed significantly (P < 0.05) among lines. Two lines from UNIPD, 6 and 9, were the most susceptible to the disease, whereas the other lines showed similar levels. The three isolates of F. oxysporum f. sp. betae differed significantly (P < 0.05) in disease severity. Five weeks after inoculation the plants were harvested and roots examined. Surprisingly, severe root rot was observed in the susceptible UNIPD lines when inoculated with all three isolates, while this symptom was never observed in the USDA germplasm. The development of this disease symptom obviously depends on the plant genotype.     

Improved control of black rot of broccoli caused by <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis> using a bacteriophage and a nonpathogenic <Emphasis Type="Italic">Xanthomonas</Emphasis> sp. strain

We examined the potential for biological control of black rot of broccoli, caused by Xanthomonas campestris pv. campestris (Xcc), using nonpathogenic Xanthomonas sp. strain 11-100-01 (npX) mixed with bacteriophage XcpSFC211 (pXS). Inoculation of intact broccoli plants in greenhouse trials with either npX or pXS did not control black rot. After injured plant inoculation, however, npX alone or npX with pXS significantly controlled black rot. When a mixed suspension of npX with pXS was placed on a membrane filter, then washed with distilled water and air-dried, a substantial amount of pXS adsorbed to the surface of npX. In a field trial, broccoli plants were sprayed with a suspension of npX with pXS, then inoculated with Xcc. A meta-analysis of the results from five field trials showed an integrated risk ratio (IRR, the ratio of disease incidence in inoculated broccoli plants to the incidence in control plants) of 0.69 after treatment with only npX and 0.59 with npX with pXS, indicating that black rot incidence was significantly reduced by each treatment. The difference between these two treatments was also significant. IRR was 1.24 when comparing suppression by npX with pXS and that by basic copper sulfate wettable powder; thus, their control was comparable. The combination of npX with pXS improved the preventive effect against black rot. This is the first report describing that a nonpathogenic Xanthomonas sp. strain mixed with a bacteriophage effectively controlled black rot of broccoli in field trials.     

AlgU contributes to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis> DC3000 by regulating production of the phytotoxin coronatine

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.     

Identification and community dynamics of fungi associated with root,crown, and foot rot of field pea in western Canada

Fungal and oomycete communities in symptomatic roots, crowns and lower stem tissues of field pea plants from sites across the Canadian prairies were characterized using plate culture and sequence-based identification. Symptomatic plants were sampled at the mid-flowering stage from a total of 21 fields in 2014 and 2015. Fungi and oomycete species were isolated on agar medium and grouped into operational taxonomic groups (OTU) based on their morphology. From the OTUs, 40 fungal and oomycete species were identified according to translation elongation factor-1 alpha or ITS sequences. Fusarium spp. accounted for more than 60 % of total isolations. The fungal communities were similar in roots and crowns, but differed from communities in lower stems. In 2014, the most prevalent fungi isolated from roots were F. avenaceum, F. solani and F. redolens, but F. solani was dominant in 2015. In crown tissues, F. avenaceum was most prevalent in 2014 but F. solani was more prevalent in 2015. Stem tissues were primarily colonized by Peyronellaea spp. (= Ascochyta / Phoma spp.) and Alternaria spp. in 2014, but there was a drastic decrease in Peyronellaea spp. in 2015. This study indicated that climate, primarily precipitation levels, and local edaphic characteristics may play a profound role in the structure of phytopathogen communities associated with the root and foot rot diseases complex of field pea on the Canadian prairies.     

Carabidae as potential biological agents for controlling infestations of the cabbage root fly

Laboratory tests with carabid ground beetles found in Wellesbourne fields indicated that intermediate-sized beetles such asBembidion tetracolum, Amara familiar is andAgonwn dorsale were more effective predators of cabbage root fly (Erioischia brassicae) eggs than small beetles, e.g.Bembidion lampros andTrechus quadristriatus, or large beetles, e.g.Harpalus rufipes andPterostichus melanarius. Further tests indicated thatA. dorsale would be an ideal predator, as it can survive up to one month without food, is not killed byP. melanarius, aggregates naturally, and eats large numbers of cabbage root fly eggs/larva.A. dorsale is also relatively easy to retain near the stems of plants by simple barriers and is not cannibalistic. Unfortunately, sufficientA. dorsale were not available for greenhouse trials. Greenhouse trials withB. tetracolum indicated that two beetles per plant were sufficient to control the high levels of cabbage root fly infestation normally encountered in the field at Wellesbourne.     

Histopathology of the infection on resistant and susceptible lentil accessions by two contrasting pathotypes of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">lentis</Emphasis>

Wilt disease of lentil caused by Fusarium oxysporum f.sp. lentis (Fol) is one of the most important diseases affecting lentil worldwide. Differential response of six lentil accessions with reported differences in the level of resistance to Fol was studied micro and macroscopically. Penetration took place through root epidermal cells without formation of any specific structure. Hyphae reached the stele within two days after inoculation (dai) and subsequently invaded xylem bundles having colonised the endodermis, vascular system and even vascular parenchyma phloem already by 4 dai. Resistance was observed as a quantitative trait in all studied accessions resulting from varying levels of xylem occlusion with gum-like substances and of degree of colonization observed only after 4 dai. An indication of a qualitative resistance was detected in accession BGE019696 inoculated with pathotype 1 as a fast secretion of phenolic compounds at 4 dai. Plasmolysis of cytoplasm, lignification and accumulation of phenolic compounds, gum-like substances and/or tyloses were observed from 15 to 30 dai. As a result of the various operative mechanisms, significantly lower numbers of propagules were recovered from roots by 15 dai, and a retardation of disease was measured as lower disease index by 30 dai in plants inoculated with pathotype 1, but not in those inoculated with pathotype 7.     

Tight regulation of allene oxide synthase (AOS) and allene oxide cyclase-3 (AOC3) promote <Emphasis Type="Italic">Arabidopsis</Emphasis> susceptibility to the root-knot nematode <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

Biosynthesis of the oxylipin jasmonic acid (JA) in Arabidopsis thaliana is catalyzed by a single allene oxide synthase (AOS)-encoding gene and four genes encoding four functional allene oxide cyclase (AOC) polypeptides (AOC1, AOC2, AOC3, and AOC4). To elucidate the biological activities of the JA pathway in regulating the plant defense response to plant-parasitic nematodes, transgenic lines carrying the GUS reporter gene under the control of individual AOC or AOS promoters were examined. Upon penetration by second-stage juveniles (J2 s), promoter activities of AOC1, AOC3 and AOC4 appeared in the root tip and root-elongation zone, with AOC3 demonstrating highest induction. At 5 days AOC3 activity continued to be highly pronounced in the stele and root cortex, associated with nematode invasion throughout gall initiation and maturation. AOS expression appeared 3 days postinfection and accompanied all later infection stages. Mutant lines were analyzed: disruption in AOS rendered plants more resistant to nematode infection, as reflected by the decreased number of females produced on this line; loss-of-function of AOC3 rendered plants more susceptible to nematode infection. Oxylipins derived from the 9- and 13-lipoxygenase pathways were assayed for their direct inhibitory activity toward M. javanica J2 s. Clear nematicidal activity of the bioactive 9- and 13-hydroperoxides was observed. Oxylipins produced by divinyl ether synthase, colneleic acid, colnelenic acid and ω5(Z)-etherolenic acid demonstrated strong inhibitory activity. These data, along with those of other assayed oxylipins, suggest that temporal and spatial fine tuning of the JA route allowing nematodes parasitism on plant host.