Effector gene screening allows unambiguous identification of Fusarium oxysporum f. sp. lycopersici races and discrimination from other formae speciales

During infection of tomato, the fungus Fusarium oxysporum f. sp. lycopersici secretes several unique proteins, called 'secreted in xylem' (Six) proteins, into the xylem sap. At least some of these proteins promote virulence towards tomato and among them, all predicted avirulence proteins that can trigger disease resistance in tomato have been found. In this study, a large, worldwide collection of F. oxysporum isolates was screened for the presence of seven SIX genes ( SIX1 – SIX7 ). The results convincingly show that identification of F. oxysporum formae speciales and races based on host-specific virulence genes can be very robust. SIX1, SIX2, SIX3 and SIX5 can be used for unambiguous identification of the forma specialis lycopersici . In addition, SIX4 can be used for the identification of race 1 strains, while polymorphisms in SIX3 can be exploited to differentiate race 2 from race 3 strains. For SIX6 and SIX7 , close homologs were found in a few other formae speciales , suggesting that these genes may play a more general role in pathogenicity. Host specificity may be determined by the unique SIX genes, possibly in combination with the absence of genes that trigger resistance in the host.     

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.     

Identification of F. o. cucumerinum and F. o. luffae by RAPD-generated DNA probes

AIMS: To create a fast, sensitive and specific method for identifying Fusarium oxysporum f. sp. cucumerinum and F. o. luffae. METHODS AND RESULTS: Specific DNA bands were selected as probes from RAPD profiles of 13 formae speciales of F. oxysporum. The forma specialis-specific probe OPC18300c and OPC18520f could be used to identify F. o. cucumerinum and F. o. luffae by RAPD-PCR followed dot blot hybridization, respectively. CONCLUSIONS: A specific method for identifying F. o. cucumerinum and F. o. luffae was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: F. oxysporum formae speciales identification with a DNA probe can be relatively rapid and provides a method to identify the pathogen without host inoculation tests.     

Characterization of the formae speciales of Fusarium oxysporum causing wilts of cucurbits by DNA fingerprinting with nuclear repetitive DNA sequences.

The genetic relatedness of five formae speciales of Fusarium oxysporum causing wilts of cucurbit plants was determined by DNA fingerprinting with the moderately repetitive DNA sequences FOLR1 to FOLR4. The four FOLR clones were chosen from a genomic library made from F. oxysporum f. sp. lagenariae 03-05118. Total DNAs from 50 strains representing five cucurbit-infecting formae speciales, cucumerinum, melonis, lagenariae, niveum, and momordicae, and 6 strains of formae speciales pathogenic to other plants were digested with EcoRV and hybridized with 32P-labeled FOLR probes. The strains were clearly distinguishable at the formae specialis level on the basis of FOLR DNA fingerprints. Fifty-two fingerprint types were detected among the 56 strains by using all FOLR probes. These probes were used to infer phylogenetic relationships among the DNA fingerprint types by the unweighted pair group method using averages and parsimony analysis. The fingerprint types detected in each of the formae speciales cucumerinum, lagenariae, niveum, and momordicae were grouped into a single cluster. However, two different genetic groups occurred in the formae specialis melonis. The two groups also differed in pathogenicity: one group caused wilts of muskmelon and oriental melon, while the second was pathogenic only to muskmelon. The fingerprint types of different formae speciales pathogenic to plants other than cucurbits were distinguishable from one another and from the fingerprints of the cucurbit-infecting strains. These results suggest that the cucurbit-infecting formae speciales are intraspecific variants distinguishable at the DNA level and in their host range.     

A highly conserved effector in Fusarium oxysporum is required for full virulence on Arabidopsis

Secreted-in-xylem (SIX) proteins of the vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici are secreted during infection of tomato and function in virulence or avirulence. F. oxysporum formae speciales have specific host ranges but the roles of SIX proteins in diverse hosts are unknown. We identified homologs of F. oxysporum f. sp. lycopersici SIX1, SIX4, SIX8, and SIX9 in the genome of Arabidopsis infecting isolate Fo5176. A SIX4 homolog (termed Fo5176-SIX4) differed from that of F. oxysporum f. sp. lycopersici (Fol-SIX4) by only two amino acids, and its expression was induced during infection of Arabidopsis. Transgenic Arabidopsis plants constitutively expressing Fo5176-SIX4 had increased disease symptoms with Fo5176. Conversely, Fo5176-SIX4 gene knock-out mutants (Δsix4) had significantly reduced virulence on Arabidopsis, and this was associated with reduced fungal biomass and host jasmonate-mediated gene expression, the latter known to be essential for host symptom development. Full virulence was restored by complementation of Δsix4 mutants with either Fo5176-SIX4 or Fol-SIX4. Thus, Fo5176-SIX4 contributes quantitatively to virulence on Arabidopsis whereas, in tomato, Fol-SIX4 acts in host specificity as both an avirulence protein and a suppressor of other race-specific resistances. The strong sequence conservation for SIX4 in F. oxysporum f. sp. lycopersici and Fo5176 suggests a recent common origin.     

Gerbera jamesonii, a New Host of Fusarium oxysporum f.sp. tracheiphilum

The random amplified polymorphic DNA (RAPD) technique was used to analyze the total genomic DNA of pathogenic isolates of Fusarium oxysporum on Gerbera jamesonii by comparing them to representatives of the formae speciales chrysanthemi and tracheiphilum. A close genetic relationship was observed among most of the new isolates from G. jamesonii. They shared RAPD markers with the tested representatives of the forma specialis chrysanthemi . Some isolates of those tested from diseased G. jamesonii were placed in a different cluster, which included representative isolates of forma specialis tracheiphilum . This is the first report of F. oxysporum f.sp. tracheiphilum on G. jamesonii. A rapid protocol for DNA extraction directly from fungal colonies grown on potato dextrose agar allowed complete analysis in less than 4 h.     

Pantothenate synthetase from Fusarium oxysporum f. sp. lycopersici is induced by alpha-tomatine

The steroidal glycoalkaloid alpha-tomatine which is present in tomato (Lycopersicum sculentum) is assumed to protect the plant against phytopathogenic fungi. We have isolated a gene from the fungal pathogen Fusarium oxysporum f. sp. lycopersici that is induced by this glycoalkaloid. This gene, designated panC, encodes a predicted protein with a molecular mass of 41 kDa that shows a high degree of sequence similarity to pantothenate synthetases from yeast, plants and bacteria. Recombinant PanC protein from F. oxysporum has been over-expressed in Escherichia coli and purified to homogeneity. It shows pantothenate synthetase activity in the presence of D-pantoate, beta-alanine and ATP. The panC gene from F. oxysporum functionally complements an E. coli panC mutant, demonstrating that the PanC protein functions in vivo as a pantothenate synthetase. Southern analysis of F. oxysporum genomic DNA from other formae speciales indicates that there is a single copy of the pantothenate syntethase gene in this fungus. The presence of a STRE consensus sequence (CCCCT) in the promoter region of the gene suggests that the induction of panC may be part of a cellular stress response triggered by alpha-tomatine.     

尖孢镰刀菌四个专化型细胞核的初步研究

本文对尖孢镰刀菌(Fusarium oxysporum)的4个专化型12株菌的孢子萌发、核分裂时间及核DNA含量进行了比较,结果表明:不同专化型菌株孢子萌发速度基本一致,而萌发过程中发生第一次核分裂的时间不同。黄瓜、西瓜、荸荠、大豆各专化型第一次核分裂时间分别为:5.67hr、5.45hr、7.35hr、7.82hr,其核DNA含量分别为:0.321pg、o.306pg、0.177pg 0.174pg。测定结果显示出不同专化型菌株的孢子核DNA含量可能存在倍数关系,并推测F.oxysporum的黄瓜专化型与西瓜专化型为同一类型,而荸荠专化型与大豆专化型为同一类型。     

利用脂肪酸生物标记对尖镰孢寄主专化型的快速鉴定

尖镰孢寄主范围广、遗传差异大,其种下存在多种寄主专化型。对尖镰孢寄主专化型的快速鉴定可为科学制定植物病害防控策略提供依据。利用Sherlock MIS脂肪酸鉴定系统对分离自番茄、棉花、黄瓜、茄子等4种寄主专化型的18株尖镰孢进行脂肪酸成分测定,共检测到10种脂肪酸。运用SPSS软件中的PCA法对被检测到的脂肪酸进行主成分分析,确定了18:1CIS9（W9）[X1],18:2 CIS 9,12/18:0a[X2]和18:00[X3]等3个脂肪酸为其主成分。利用Bayes逐步判别法建立了尖镰孢4种不同寄主专化型判别模型为Y1=-157.750+2.809X1+3.391X2+8.099X3;Y2=-178.343+0.586X1+7.587X2- 0.214X3;Y3=-129.132+2.749X1+4.163X2+4.476X3;Y4=-201.307+2.016X1+7.345X2+2.400X3。通过对43株未知寄主专化型菌株主成分脂肪酸的测定,利用判别法对尖镰孢进行判定,结果发现有40株与原寄主来源一致,判对率达93%。表明脂肪酸生物标记法可用于尖镰孢寄主专化型的快速鉴定。     

Isozyme Variation and Genetic Distances of Erysiphe graminis DC. Formae Speciales

Abstract Isozymes of ten different enzymes and unspecific stained proteins were used as biochemical genetic markers to study genetic variation within and between E. graminis ff. sp. hordei, avenae, secalis and tritici. In addition, grainproteins of the corresponding host species were examined. In each forma specialis, one genotype proved to be predominant. 131 distinct isozyme and 93 protein bands were distinguishable in these genotypes. However, divergent banding patterns differed only in 8 bands from the predominant banding patterns found within the formae speciales avena, secalis and tritici. The genetic relationships between powdery mildew formae speciales and host species were computed by cluster analysis from similarity (F) and dissimilarity (D) coefficients and illustrated by phylogenetic trees. Marked correspondence was found between E. graminis ff. sp. secalis and tritici (F: 82–90%). Lower homologies were obtained from the comparison ofthese formae speciales respectively with E. graminis ff. sp. hordei (F: 28–34%) and avenae (F: 24–32%). All phylogenetic trees constructed revealed the same arrangement classification of the formae speciales with similar graduation. The comparison of the host species revealed the highest similarity between S. cereale and T. aestivum (F: 74%). Regression analysis confirmed significant correlation between the genetic relationships within host species and powdery mildew formae speciales (r²= 0.81).     

应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells

Fusarium oxysporum is an asexual, soil inhabiting fungus that comprises many different formae speciales, each pathogenic towards a different host plant. In absence of a suitable host all F. oxysporum isolates appear to have a very similar lifestyle, feeding on plant debris and colonizing the rhizosphere of living plants. Upon infection F. oxysporum switches from a saprophytic to an infectious lifestyle, which probably includes the reprogramming of gene expression. In this work we show that the expression of the known effector gene SIX1 of F. oxysporum f. sp. lycopersici is strongly upregulated during colonization of the host plant. Using GFP (green fluorescent protein) as reporter, we show that induction of SIX1 expression starts immediately upon penetration of the root cortex. Induction requires living plant cells, but is not host specific and does not depend on morphological features of roots, since plant cells in culture can also induce SIX1 expression. Taken together, F. oxysporum seems to be able to distinguish between living and dead plant material, preventing unnecessary switches from a saprophytic to an infectious lifestyle.     

In Vitro Physiological Responses of Fusarium oxysporum f. sp. niveum to Exogenously Applied Syringic Acid

ABSTRACT. Plant–microbe interactions are often accompanied by allelochemicals, such as syringic acid, released from the host plant. To explore the role of phenolic acids released from crop host plants in response to pathogen invasion, we examined the allelopathic effect of an artificially applied syringic acid on Fusarium oxysporum f. sp. niveum . We demonstrated that the growth and the conidial germination rate of F. oxysporum f. sp. niveum were stimulated at lower concentrations of syringic acid, though inhibited by higher dosage compared with control. The yield of fungus mycotoxin was increased from 60.9% to 561.5%. We conclude that syringic acid can be considered as a allelochemical inducer, stimulating the relative virulence factors of invading pathogens.     

Characterization of the gene encoding pisatin demethylase (FoPDA1) in Fusarium oxysporum

The pea pathogen Fusarium oxysporum f. sp. pisi is able to detoxify pisatin produced as a defense response by pea, and the gene encoding this detoxification mechanism, FoPDA1, was 82% identical to the cytochrome P450 pisatin demethylase PDA1 gene in Nectria haematococca. A survey of F. oxysporum f. sp. pisi isolates demonstrated that, as in N. haematococca, the PDA gene of F. oxysporum f. sp. pisi is generally located on a small chromosome. In N. haematococca, PDA1 is in a cluster of pea pathogenicity (PEP) genes. Homologs of these PEP genes also were found in the F. oxysporum f. sp. pisi isolates, and PEP1 and PEP5 were sometimes located on the same small chromosomes as the FoPDA1 homologs. Transforming FoPDA1 into a pda(?) F. oxysporum f. sp. lini isolate conferred pda activity and promoted pathogenicity on pea to some transformants. Different hybridization patterns of FoPDA1 were found in F. oxysporum f. sp. pisi but these did not correlate with the races of the fungus, suggesting that races within this forma specialis arose independently of FoPDA1. FoPDA1 also was present in the formae speciales lini, glycines, and dianthi of F. oxysporum but they had mutations resulting in nonfunctional proteins. However, an active FoPDA1 was present in F. oxysporum f. sp. phaseoli and it was virulent on pea. Despite their evolutionary distance, the amino acid sequences of FoPDA1 of F. oxysporum f. sp. pisi and F. oxysporum f. sp. phaseoli revealed only six amino acid differences, consistent with a horizontal gene transfer event accounting for the origin of these genes.     

Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-alpha1,4-polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.