Endopolygalacturonase Gene Research Articles

Efficient Expression of an Acidic Endo-polygalacturonase from Aspergillus niger and Its Application in Juice Production.

An endo-polygalacturonase gene (pga-zj5a) was cloned by reverse transcription from cDNAs synthesized from Aspergillus niger ZJ5 total RNA. The open reading frame of pga-zj5a was 1089 base pairs encoding 362 amino acids. Pga-zj5a lacking a signal peptide sequence was successfully amplified using A. niger ZJ5 cDNA as the template and was ligated into the pPIC9 vector. The resulting plasmid was transformed into competent cells of Pichia pastoris GS115 for heterologous expression. The polygalacturonase showed a maximum activity level of 10436 U/mL in the culture supernatant from a 3 L fermenter. Assays of enzymatic properties showed that the optimal pH and temperature of the recombinant PGA-ZJ5A were 4.5 and 40 °C, respectively. PGA-ZJ5A was effective in pear juice clarification, increased the volume of pear juice by 41.8%, and improved its light transmittance 3-fold.

The Constitutive Endopolygalacturonase TvPG2 Regulates the Induction of Plant Systemic Resistance by Trichoderma virens.

Trichoderma spp. are opportunistic fungi some of which are commonly present in the rhizosphere. Several species, such as T. virens, are also efficient biocontrol agents against phytopathogenic fungi and exert beneficial effects on plants. These effects are the consequence of interactions between Trichoderma and plant roots, which trigger enhanced plant growth and induce plant resistance. We have previously shown that T. virens I10 expresses two endopolygalacturonase genes, tvpg1 and tvpg2, during the interaction with plant roots; tvpg1 is inducible while tvpg2 is constitutively transcribed. Using the same system, the tomato polygalacturonase-inhibitor gene Lepgip1 was induced at the same time as tvpg1. Here we show by gene disruption that TvPG2 performs a regulatory role on the inducible tvpg1 gene and in triggering the plant immune response. A tvpg2-knockout strain fails to transcribe the inducible tvpg1 gene in neither in vitro in inducing media containing pectin or plant cell walls, nor during the in vivo interaction with tomato roots. Likewise, the in vivo induction of Lepgip1 does not occur, and its defense against the pathogen Botrytis cinerea is significantly reduced. Our data prove the importance of a T. virens constitutively produced endopolygalacturonase in eliciting plant induced systemic resistance against pathogenic fungi.

Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production

Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants Km and Vmax of PoxaEnPG28A were calculated as 1.57g/L and 14,641.29U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.

First Report of Alternaria alternata Causing Hybrid Hazel (Corylus heterophylla × C. avellana) Fruit Drop in China

HomePlant DiseaseVol. 101, No. 2First Report of Alternaria alternata Causing Hybrid Hazel (Corylus heterophylla × C. avellana) Fruit Drop in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Alternaria alternata Causing Hybrid Hazel (Corylus heterophylla × C. avellana) Fruit Drop in ChinaY. Q. Cheng, Y. T. Ming, B. W. Yang, T. Liu, and J. F. LiuY. Q. ChengSearch for more papers by this author, Y. T. MingSearch for more papers by this author, B. W. YangSearch for more papers by this author, T. LiuSearch for more papers by this author, and J. F. LiuSearch for more papers by this authorAffiliationsAuthors and Affiliations Y. Q. Cheng Y. T. Ming B. W. Yang T. Liu J. F. Liu , Jilin Provincial Key Laboratory of Plant Resource Science and Green Production, Jilin Normal University, Siping, Jilin 136000, China. Published Online:14 Nov 2016https://doi.org/10.1094/PDIS-07-16-1079-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Previously undocumented apical necrosis has been observed in hybrid hazels (Corylus heterophylla × C. avellana) since 2012, causing premature fruit drop rates of up to 60% and leading to major economic losses in China. Hazelnut apical necrosis is also an important production problem in Italy (Belisario et al. 2004; Santori et al. 2010). To explore the cause of hazelnut apical necrosis, 352 isolates were extracted from 150 fruits with external symptoms. Fruits were surface-disinfected, rinsed in sterile water, plated, and incubated for 1 week (25°C, 16/8 h light/dark), after a June to August 2015 collection from trees and below-canopy ground at five hybrid-hazel orchards in Jilin Province (Belisario et al. 2004). The 14 most common, morphologically distinct isolates were used for pathogenicity evaluation on immature hazelnut: Alternaria spp. (A01-A05), Fusarium spp. (F01-F03), Penicillium spp. (P01-03), Trichothecium roseum (Tr01), Aspergillus niger (As01), and Rhizopus stolonifer (Rs01). Ten fruit samples per isolate were inoculated with 50 μl (1 × 106 conidia/ml). Three replicates were conducted. Only one isolate (A01) was pathogenic, causing external symptoms on the fifth day post inoculation (e.g., apical necrotic lesions of 2 to 5 mm in diameter that grew to 10 to 20 mm 10 days post inoculation). The other 13 isolates produced superficial brown-blackish necrosis around the inoculation point only 20 to 30 days post inoculation; they grew on dead tissue and covered fruit with mycelium only after necrosis developed from the inoculation wound. A01-inoculated tissues showing typical apical-necrosis symptoms were dissected for in situ microscopic morphological examination. A01 exhibited septate mycelia with conidiophores, and its size averaged 25.2 × 8.48 μm based on measurements of 100 conidia. Conidia were dark with an ovoid shape, typically possessing both cross and longitudinal septa. In addition to morphological characteristics, the pathogen was confirmed as A. alternata based on PCR amplification and sequencing of rDNA ITS, endoPG (endopolygalacturonase gene), and OPA1-3 (anonymous region of A. alternata genome) (Peever et al. 2004). Sequencing results (GenBank accession nos. KU9865082, KX618660, and KX618659) yielded 571 bp of rDNA ITS, 460 bp of endoPG, and 805 bp of OPA1-3. BLASTn analysis revealed that these sequences were 100% identical to A. alternata isolates (KJ082099.1, KU9865082, and KR493348.1). As confirmation of A01 virulence, 100 immature fruit samples were inoculated with 50 μl conidial suspension (1 × 106 conidia per ml of H2O). Double-distilled water was used as a control (100 samples). Apical necrosis symptoms appeared on inoculated fruits, but not on controls, 10 days post inoculation. Isolated tissue was necrotic, blackish, and led to fruit drop. An isolate identical to A01 was extracted from diseased tissues. This is the first report linking A. alternata to an internal form of hazelnut apical necrosis in China.

Detection of cabbage yellows fungus Fusarium oxysporum f. sp. conglutinans in soil by PCR and real-time PCR

For the specific detection of Fusarium oxysporum f. sp. conglutinans (Foc), we designed a primer set for PCR and a primer–probe set for real-time PCR based on its endopolygalacturonase gene sequence. Using the primer set, we could distinguish Foc from other pathogenic forms and nonpathogenic isolates of F. oxysporum. Moreover, we detected the fungus by real-time PCR using DNA isolated from soil. Sensitivity of real-time PCR was improved 10- to 10,000-fold by adding bovine serum albumin or by performing pre-PCR. Our method can detect Foc in the soil at a density that causes only slight yellows symptom.

Cloning and Expression of Pectobacterium carotovorum Endo-polygalacturonase Gene in Pichia pastoris for Production of Oligogalacturonates

A bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum was cloned into pGAPZαA vector and constitutively expressed in Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The enzyme had optimum activity at pH 5.5 and 40 °C with stability between pH 5.0 and 8.0 and at temperatures up to 50 °C. The enzyme activity was enhanced by 41% with the addition of 1 mM Co++, and inhibited by Fe++ with a 63% reduction. The mode of the enzyme action showed internal cleavage of α-1,4 glycoside bonds of polygalacturonic acid and citrus peel pectin. Trigalacturonate and hexagalacturonate were the main hydrolysis products, with a yield of 0.44±0.01 and 0.21±0.01 mg released per mg polygalacturonic acid substrate, respectively. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products.

A New Accurate Genotyping HRM Method for Alternaria Species Related to Fruit Rot Diseases of Apple and Pomegranate

Alternaria core rot and Alternaria black heart rot of apple and pomegranate fruit, respectively, are major pre- and postharvest diseases worldwide. However, it is very difficult to differentiate the rot related Alternaria species in the Alternaria complex as they are not always correlate to species-groups based upon morphological characteristics and due to the limited genetic variation these species exhibit among each other. Therefore, it is crucial to exploit novel assays towards the accurate identification and differentiation of these Alternaria species. We have developed, a real-time PCR assay [using species specific primers targeting the endopolygalacturonase (EndoPG) gene] combined with a high-resolution melting (HRM) curve analysis for discrimination of the 14 single nucleotide polymorphisms (SNPs)-based Alternaria haplotypes, which were assigned based on the aligned sequence profiles of 138 Alternaria spp. strains previously isolated from apple and pomegranate rotted fruit. This analysis specifically generated 14 unique HRM curve haplotype profiles among the Alternaria complex species tested. The results showed that HRM curve analysis allows the rapid and adequate identification and genotyping of the three Alternaria species (A. alternata, A. tenuissima and A. arborescens) responsible mostly for the apple and pomegranate fruit rot diseases.

In planta expression profiles and sequence variation of the endopolygalacturonase gene in Venturia nashicola, the causal agent of Asian pear scab

Venturia nashicola, causing scab disease on Japanese and Chinese pears, is one of the most devastating pathogens of these crops. Previous studies suggested the potential role of endopolygalacturonase (endoPG) during the invasion stage and sequence variations of the endoPG gene were thought to be effective for pathological race identification. To reveal the role of endoPG during the infection stage, the expression profiles of the endoPG gene of V. nashicola were examined in susceptible and resistant pear cultivars. In the susceptible cv. Kousui, although the biomass of V. nashicola race 1 isolate increased after inoculation, the relative level of endoPG expression decreased obviously. By contrast, in the race-specific resistant pear strain Mamenashi 12, the expression of endoPG increased significantly until 3 dpi though the biomass fluctuated transiently after inoculation. These results indicated that endoPG may not be a crucial factor in the pathogenicity of V. nashicola race 1 isolates when infecting cv. Kousui. For the second purpose, sequence variations of the endoPG gene from different pathological races were not race-specific, and these sequence variations could not be used for race identification. However, endoPG phylogeny supported the separation of V. nashicola from V. pirina, the cause of scab on European pear.

Yeast Kluyveromyces lactis as host for expression of the bacterial lipase: cloning and adaptation of the new lipase gene from Serratia sp.

Many microbial lipases have been successfully expressed in yeasts, but not in industrially attractive Kluyveromyces lactis, which among other benefits can be cultivated on a medium supplemented with whey--cheap and easily available industrial waste. A new bacterial lipase from Serratia sp. was isolated and for the first time expressed into the yeast Kluyveromyces lactis by heterologous protein expression system based on a strong promoter of Kluyveromyces marxianus triosephosphate isomerase gene and signal peptide of Kluyveromyces marxianus endopolygalacturonase gene. In addition, the bacterial lipase gene was synthesized de novo by taking into account a codon usage bias optimal for K. lactis and was expressed into the yeast K. lactis also. Both resulting strains were characterized by high output level of the target protein secreted extracellularly. Secreted lipases were characterized for activity and stability.

Expression of Five Endopolygalacturonase Genes and Demonstration that MfPG1 Overexpression Diminishes Virulence in the Brown Rot Pathogen Monilinia fructicola.

Monilinia fructicola is a devastating pathogen on stone fruits, causing blossom blight and fruit rot. Little is known about pathogenic mechanisms in M. fructicola and related Monilinia species. In this study, five endopolygalacturonase (endo-PG) genes were cloned and functionally characterized in M. fructicola. Quantitative reverse-transcriptase PCR (qRT-PCR) revealed that the five MfPG genes are differentially expressed during pathogenesis and in culture under various pH regimes and carbon and nitrogen sources. MfPG1 encodes the major endo-PG and is expressed to significantly higher levels compared to the other four MfPGs in culture and in planta. MfPG1 function during pathogenesis was evaluated by examining the disease phenotypes and gene expression patterns in M. fructicola MfPG1-overexpressing strains and in strains carrying the β-glucuronidase (GUS) reporter gene fused with MfPG1 (MfPG1-GUS). The MFPG1-GUS reporter was expressed in situ in conidia and hyphae following inoculation of flower petals, and qRT-PCR analysis confirmed MfPG1 expression during pathogenesis. MfPG1-overexpressing strains produced smaller lesions and higher levels of reactive oxygen species (ROS) on the petals of peach and rose flowers than the wild-type strain, suggesting that MfPG1 affecting fungal virulence might be in part resulted from the increase of ROS in the Prunus–M. fructicola interactions.

Two Endopolygalacturonase Genes in Trichoderma virens: In Silico Characterization and Expression during Interaction with Plants

AbstractThe ability of Trichoderma spp. to antagonize most phytopathogenic fungi and to interact with plants by inducing their resistance and promoting their growth has been widely described. Endopolygalacturonases (endoPGs) produced by Trichoderma spp. can assist root penetration and play a pre‐eliciting role in induced systemic resistance (ISR), a beneficial effect detected in plants colonized by Trichoderma. In this study, two endoPG genes (Tvpg1 and Tvpg2) have been identified and partially characterized in a T. virens isolate, previously investigated for its antagonistic ability in several biological systems. An extensive in silico analysis showed relatively low sequence similarities (53%) between TvPG1 and TvPG2 proteins and underscored interesting phylogenetic relationships in the comparison of endoPG genes among fungal genomes. The phylogeny of endoPGs suggests a differentiation in action mechanisms associated with biological functions as one cluster shows only genes from plant‐interacting organisms. A gene expression analysis demonstrated a different regulation pattern for those genes. The Tvpg1 gene was induced both when the fungus was grown in liquid cultures supplemented with pectin or plant cell walls and when it was applied to tomato roots in a growth chamber. Expression times were comparable in both systems. On the contrary, the Tvpg2 gene displayed a constitutive expression in all conditions tested, including controls. The molecular communication between tomato roots and T. virens was investigated by checking the expression of a tomato PGIP (Lepgip1) in roots colonized by fungal mycelium. The Lepgip1 transcript was induced at times coincident with Tvpg1 expression, suggesting a correlation between the two gene products. This paper reports the results of first investigations of T. virens endoPG genes performed on a structural, phylogenetic and functional level.

Secretory Expression and Characterization of an Acidic Endo-Polygalacturonase Gene from Aspergillus niger SC323 in Saccharomyces cerevisiae.

The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The full-length cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50°C, respectively. The Michaelis-Menten constant (Km) and maximal velocity (Vmax) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca(2+), Cu(2+), and Na(+), and strongly inhibited by Pb(2+) and Mn(2+). The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.

Single nucleotide polymorphism analysis of the endopolygalacturonase gene in peach and its potential use in crossbreeding programs.

Single nucleotide polymorphisms (SNPs) are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in genetic diversity studies and crossbreeding programs. In this study, we examined 113 DNA sequences of the endopolygalacturonase (endo-PG) gene from 67 peach accessions and found a total of 56 SNPs and 6 insertion/deletions (indels), with a frequency of 3, 1, and 3% for the transitions, transversions, and indels, respectively. Meanwhile, the majority of the observed SNPs were found in the intron regions, while only 2 variable sites and a single indel were detected in the exon regions. A dendrogram was obtained using neighbor-joining cluster analysis and divided into 2 main groups, providing evidence that most of the accessions of the clingstone nonmelting flesh phenotypes generally clustered together and were comparatively nonrelated to the "stony hard" peach cultivars, which were in a different branch altogether. Furthermore, 4 major haplotypes were formed and 3 cleaved amplified polymorphic sequence primer sets were mined according to fruit texture and stone adhesion, displaying their potential as candidate molecular markers for discriminating genotypes. This research will assist peach genetic enhancement by introducing a novel crossbreeding strategy.

Molecular characterization of a thermophilic endo-polygalacturonase from Thielavia arenaria XZ7 with high catalytic efficiency and application potential in the food and feed industries.

Thermophilic endo-polygalacturonases with high catalytic efficiency are of great interest in the food and feed industries. This study identified an endo-polygalacturonase gene (pg7fn) of glycoside hydrolase family 28 in the thermophilic fungus Thielavia arenaria XZ7. Recombinant PG7fn produced in Pichia pastoris is distinguished from other enzyme counterparts by its high functional temperature (60 °C) and specific activity (34382 ± 351 U/mg toward polygalacturonic acid). The enzyme exhibited good pH stability (pH 3.0-8.0) and resistance to pepsin and trypsin digestion and had a significant effect on disaggregation of soybean meal. Addition of 1 U/g PG7fn increased the pectin bioavailability by 19.33%. The excellent properties described above make PG7fn valuable for applications in the food and feed industries. Furthermore, a comparative study showed that N-glycosylation improved the thermostability and catalytic efficiency of PG7fn.

Identification, characterization and mycotoxigenic ability of Alternaria spp. causing core rot of apple fruit in Greece

Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products.

Identification of a novel phylogenetic lineage of Alternaria alternata causing citrus brown spot in China

Alternaria alternata sensu lato, casual agent of citrus brown spot, first identified in Yunnan province in 2010 and subsequently found in Zhejiang, Hunan, Guangdong provinces, Chongqing municipality andGuangxi autonomous region in China. During 2010–2012, 86 isolates were collected from diseased citrus, of which 85 % isolates were pathogenic to Ponkan tangerine. Phylogenetic analyses of Chinese and worldwide isolates using partial sequences of an endopolygalacturonase gene (endoPG) and combined dataset ofendoPG and two anonymous loci (OPA1-3, OPA2-1) found that Chinese isolates fell into two of three previously described clades. One clade (‘clade 3’) contained isolates from Turkey and Israel, and the other clade (‘clade 1’) contained isolates from Florida, USA. None of the isolates from China fell into the last previously described clade (‘clade 2’). However, 24 isolates from Hunan, Guangdong and Guangxi fell into a fourth clade (‘clade 4’) not previously reported to be associated with citrus brown spot. This clade included multilocus haplotypes known to infect Japanese pear and strawberry. The observation that Chinese brown spot isolates fell into only two of three known worldwide lineages suggests that this fungus may not have co-evolved with its host in China but elsewhere in Southeast Asia and introduced to China.

Genetic diversity and virulence of Italian strains of Fusarium oxysporum isolated from Eustoma grandiflorum

Twenty-seven isolates of Fusarium oxysporum f. sp. eustomae obtained from diseased Eustoma grandiflorum (lisianthus) plants in north Italy, showing typical fusarium wilt symptoms, were studied for genetic diversity, pathogenicity, and vegetative compatibility (VC). The strains were examined for differences in the nucleotide sequence of translation elongation factor 1-α (tef) and two endopolygalacturonase genes (pg1 and pg5). The phylogenetic analysis performed on the isolates enabled the identification of three main groups, named I, II, and III, and two subgroups (Ia and Ib). In pg1 and pg5 sequences, most of nucleotide variations resulted in aminoacid variations, while in tef most of SNPs are in the introns or are silent. By considering the virulence of the strains included in Group I, a low virulence could be observed, Group II and III showed a reduced virulence at 30 °C compared to 20 °C. Some cultivars showed higher tolerance at 30 °C. Principal component analysis (PCA) performed on pathogenicity values, confirmed the three molecular clusters. VC identified two VCGs (vegetative compatibility groups) in Group II, one VCG in Group III, and vegetatively incompatible isolates grouped together in Group I. The integrated analysis confirmed the presence of virulence variation and genetic diversity among the F. oxysporum f. sp. eustomae isolates studied.

Discord between morphological and phylogenetic species boundaries: incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen.

BackgroundTraditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used; however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common.ResultsCoalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia.ConclusionsThe number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.

A new forma specialis of Fusarium oxysporum on Crassula ovata.

Symptoms of wilting were observed in Crassula ovata cvs Mini and Magical tree in northern Italy during summer 2010. Fourteen fungal isolates recovered from diseased tissues were analyzed by ITS sequences and identified as Fusarium oxysporum. For pathogenicity assays, roots of both cultivars plants were dipped into a conidial suspension. Inoculated plants showed typical symptoms of Fusarium wilt, confirming the virulence of all isolates. For phylogenetic analysis three genomic regions [endopolygalacturonase gene (pg5), exopolygalacturonase gene (pgx1) and Mat alpha (Mat 1-1)] were amplified by PCR, sequences were aligned with those of other formae speciales of Fusarium oxysporum from GenBank and used for constructing phylogenetic trees. Regardless of the sequenced region Crassula ovata isolates grouped together in a cluster clearly separated from other known formae speciales. Recently, a wilt disease of Cactaceae caused by F. oxysporum f. sp. opuntiarum has been reported from Italy, whose presence among the C. ovata isolates was checked by sequencing the elongation factor EF1a. The occurrence of several gaps in the sequence alignment excluded the presence of F. oxysporum f. sp. opuntiarum. Based on the results of biological and molecular investigations, it seems plausible to conclude that the F. oxysporum infecting C. ovata plants represents a novel forma specialis, for which the name F. oxysporum f. sp. crassulae, f. sp. nov. is proposed.

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.