Genetic diversity analysis and development of SCAR marker for detection of Indian populations of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis> causing chickpea wilt

Genetic diversity of the isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from 12 states representing different agro-ecological regions of India was determined through randomly amplified polymorphic DNA (RAPD) markers. The UPGMA cluster analysis grouped the isolates into eight categories showing high magnitude of genetic diversity. Each group had the isolates from different states present in various agro-ecological regions of India. Therefore, the groups generated through the RAPD analysis were not corresponding to area of the origin of the isolates. The RAPD primers, namely, OPA 7 and OPA 11 produced Foc specific fragment of ≈1.3 kb and ≈1.4 kb, respectively in all the isolates. These fragments were eluted, purified, cloned in pGEM-T Easy vector and sequenced. Primers were designed with sequence information of these two fragments using primer.3 software. Two sets of sequence characterized amplified region markers (SC-FOC 1 and SC-FOC 2) developed from the sequences of these fragments were found to be specific to Foc and produced an amplicon of 1.3 and 1.4 kb, respectively. These set of markers were validated against the isolates of the pathogen collected from different locations of India representing various races of the pathogen. They are non-specific to the other Fusarium species, Rhizoctonia solani and R. bataticola.     

Impact of biological control agents on fusaric acid secreted from <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder and Hansen in <Emphasis Type="Italic">Gladiolus grandiflorus</Emphasis> corms

Fusaric acid (FA) (5-n-butylpuridine 2-carboxyl acid), a highly toxic secondary metabolite produced by Fusarium oxysporum strains, plays a significant role in disease development. The abilities of three F. oxysporum f. sp. gladioli (Massey) Snyder and Hansen isolates (G010; 649-91; and 160-57) to produce FA in infected Gladiolus corm tissues was evaluated in vitro in relation to the presence of two biological control agents, Trichoderma harzianum T22, and Aneurinobacillus migulanus. Pathogenicity tests were used to differentiate between the abilities of the F. oxysporum strains to secrete FA. FA was identified using LC/MS and quantified using HPLC. Isolate G010 was significantly more virulent (P < 0.01) on Gladiolus grandiflorus corms; it secretes 1.8 μM FA/g fresh weight corm into inoculated Gladiolus. Moreover, G010 was the only isolate that produced FA among the three examined isolates. There was a correlation between the corm lesion area and the FA secretion ability of F. oxysporum f. sp. gladioli (P < 0.001; r ² = 0.96). No FA was detected in PDA cultures of F.oxysporum f. sp. gladioli isolates. The presence of T. harzianum T22 appeared to prevent FA secretion into the corms. In the presence of A. migulanus, however, the amount of FA secreted into the corm tissues increased. These results support the use of T. harzianum as an effective biological control agent against F. oxysporum f. sp. gladioli.     

Identification of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> in maize in northeastern China

Bremia lactucae Regel (Chromista, Peronosporaceae) is an economically destructive pathogen, which causes downy mildew disease on lettuce (Lactuca sativa L.) worldwide. The ribosomal internal transcribed spacer (ITS) of Bremia lactucae isolates was analyzed for the first time. The ITS region of lettuce downy mildew was observed to have a size of 2458 bp; thereby, having one of the longest ITS sizes recorded to date. The majority of the extremely large sized ITS2 length of 2086 was attributed to the additional presences of nine repetitive elements with lengths of 179–194 bp, which between them shared the low homology of 48–69%. Comparison of the ITS2 sequences with the B. lactucae isolates from other host plants showed that isolates present on Lactuca sativa were distinct from those on L. indica var. laciniata, as well as Hemistepta and Youngia. We suggest the high degree of sequence heterogeneity exhibited in the ITS2 region of B. lactucae may warrant the specific detection and diagnosis of this destructive pathogen or its division into several distinct species.     

Quick and accurate detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">carthami</Emphasis> in host tissue and soil using conventional and real-time PCR assay

Safflower wilt, caused by Fusarium oxysporum f. sp. carthami (Foc) is a major limiting factor for safflower (Carthamus tinctorius) production worldwide. In India alone, about 40–80% disease incidence has been reported. A rapid, efficient, specific, and sensitive diagnostic technique for Foc is therefore crucial to manage Fusarium wilt of safflower. Twenty-five isolates of F. oxysporum formae speciales infecting other crops, 17 isolates of Fusarium spp. and seven isolates of other fungal pathogens of safflower along with 75 Foc isolates were used for identification of band specific to Foc using inter-simple sequence repeat (ISSR) analysis. Out of 70 ISSR primers, the one that specifically amplified a 490 bp fragment from all the Foc isolates was selected. Sequence of the amplified fragment was utilized to design sequence characterized amplified region (SCAR) primers (FocScF/FocScR). The primer pair unambiguously and exclusively amplified a DNA fragment of approximately 213 bp in all the 75 Foc isolates. The primer set was able to detect as low as 10 pg of Foc genomic DNA using conventional PCR, while the SCAR primers when coupled with real-time qPCR demonstrated detection limits of 1 pg for Foc genomic DNA and 1000 conidia/g for soil. The assay enabled reliable diagnosis of Foc DNA in contaminated safflower fields and expedited Foc detection at 72 h post inoculation in asymptomatic seedlings. This method facilitates quick and precise detection of Foc in plant and soil samples and can be exploited for timely surveillance and sustainable management of the disease.     

RAPD based genetic diversity among different isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp<Emphasis Type="Italic">. lycopersici</Emphasis> and their comparative biocontrol

Fusarium wilt of tomato (Solanum lycopersicum Mill.) caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) W. C. Snyder and H. N. Hans (Fol.), is most serious and versatile pathogen. Chemical control of disease is not satisfactory and biological control is an attractive and potential alternative to the use of chemicals to control fusarium wilt of tomato. No any bioagent is universally effective everywhere therefore, search for potential biocontrol agent is continuous process and mandatory for several and individual ecological niches. In this experiment biocontrol efficacy of five species of Aspergillus and five species of Trichoderma were evaluated in vitro against Fusarium oxysporum f. sp. lycopersici. In both the experiments (dual culture and culture filtrates) T. harzianum was found to be highly effective against the isolates of Fol. followed by A. niger biocontrol potential of A. terreus is least among all the isolates tested. Culture filtrates obtained from A. luchuensis exerted least inhibition of Fol. The most sensitive isolate of Fol. against all the antagonists tested was identified as IIVR-2 (Fol. 9). Inherent diversity among Fol. isolates, from different tomato growing regions in India, was determined using RAPD primers. The genetic similarity coefficients ranged from 0.20 to 0.96, indicating that no any two or more isolates were 100% similar. RAPD profiles revealed up to 20% genetic diversity among ten isolates of Fusarium oxysporum f. sp. lycopersici.     

Cryptic species in the <Emphasis Type="Italic">Terfezia boudieri</Emphasis> complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5′ end of the ribosomal large subunit gene, a chitin synthase partial sequence, β-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

Fine mapping of the tomato<Emphasis Type="Italic"> I-3</Emphasis> gene for fusarium wilt resistance and elimination of a co-segregating resistance gene analogue as a candidate for<Emphasis Type="Italic"> I-3</Emphasis>

The I-3 gene from the wild tomato species Lycopersicon pennellii confers resistance to race 3 of the devastating vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici. As an initial step in a positional cloning strategy for the isolation of I-3, we converted restriction fragment length polymorphism and conserved orthologue set markers, known genes and a resistance gene analogue (RGA) mapping to the I-3 region into PCR-based sequence characterised amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers. Additional PCR-based markers in the I-3 region were generated using the randomly amplified DNA fingerprinting (RAF) technique. SCAR, CAPS and RAF markers were used for high-resolution mapping around the I-3 locus. The I-3 gene was localised to a 0.3-cM region containing a RAF marker, eO6, and an RGA, RGA332. RGA332 was cloned and found to correspond to a putative pseudogene with at least two loss-of-function mutations. The predicted pseudogene belongs to the Toll interleukin-1 receptor-nucleotide-binding site-leucine-rich-repeat sub-class of plant disease resistance genes. Despite the presence of two RGA332 homologues in L. esculentum, DNA gel blot and PCR analysis suggests that no other homologues are present in lines carrying I-3 that could be alternative candidates for the gene.Communicated by R. Hagemann     

Molecular mapping of<Emphasis Type="Italic"> Fusarium oxysporum</Emphasis> f. sp.<Emphasis Type="Italic"> ciceris</Emphasis> race 3 resistance gene in chickpea

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F₇ recombinant inbred lines derived from the cross of WR-315 (resistant) × C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h ₁ ) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.Communicated by H.C. Becker     

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">cubense</Emphasis> race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.     

Biochemical and genetic analysis of a unique poly(ADP-ribosyl) glycohydrolase (PARG) of the pathogenic fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

The genome sequence of the plant pathogen Fusarium oxysporum f. sp. lycopersici contains a single gene encoding a predicted poly(ADP-ribose) glycohydrolase (FOXG_05947.2, PARG). Here, we assessed whether this gene has a role as a global regulator of DNA repair or in virulence as an ADP ribosylating toxin homologue of bacteria. The PARG protein was purified after expressing its encoding gene in Escherichia coli. Its inhibition by 6,9-diamino-2-ethoxyacridine lactate monohydrate and tannins was similar to its human orthologue that is involved in DNA repair. A deletion strain of F. oxysporum f. sp. lycopersici showed no growth defects and was not affected in pathogenicity. Together, our results indicate that the PARG protein of F. oxysporum f. sp. lycopersici is involved in DNA repair and does not act in pathogenicity as an effector.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Physiological and biochemical responses of <Emphasis Type="Italic">in vitro Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis> to benzoic acid

The allelopathic potential of an artificially applied allelochemical, benzoic acid, on in vitro Fusarium oxysporum f.sp. niveum (a soil-borne pathogen causing watermelon wilt) was evaluated. Benzoic acid strongly inhibited its growth, sporulation and conidia germination, whereas it stimulated virulence factors of this pathogen. The biomass was reduced by 83–96 % and the conidia germinating rate and conidia production rate were decreased by 100 % at a concentration of >200 mg/L. However, phytopathogenic enzyme activities and mycotoxin production were stimulated with an increase of 10.2–1250 % for enzyme activities and 610–2630 % for mycotoxin yield.     

The development of ISSR-derived SCAR markers around the<Emphasis Type="Italic"> SEASONAL FLOWERING LOCUS</Emphasis> (<Emphasis Type="Italic">SFL</Emphasis>) in<Emphasis Type="Italic"> Fragaria vesca</Emphasis>

Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS (SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1–SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca × F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.Communicated by H. Nybom     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

<Emphasis Type="Italic">Xanthium italicum</Emphasis>, <Emphasis Type="Italic">Xanthium strumarium</Emphasis> and <Emphasis Type="Italic">Arctium lappa</Emphasis> as new hosts for <Emphasis Type="Italic">Diaporthe helianthi</Emphasis>

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.