Large‐scale molecular genetic analysis in plant‐pathogenic fungi: a decade of genome‐wide functional analysis

Plant‐pathogenic fungi cause diseases to all major crop plants world‐wide and threaten global food security. Underpinning fungal diseases are virulence genes facilitating plant host colonization that often marks pathogenesis and crop failures, as well as an increase in staple food prices. Fungal molecular genetics is therefore the cornerstone to the sustainable prevention of disease outbreaks. Pathogenicity studies using mutant collections provide immense function‐based information regarding virulence genes of economically relevant fungi. These collections are rich in potential targets for existing and new biological control agents. They contribute to host resistance breeding against fungal pathogens and are instrumental in searching for novel resistance genes through the identification of fungal effectors. Therefore, functional analyses of mutant collections propel gene discovery and characterization, and may be incorporated into disease management strategies. In the light of these attributes, mutant collections enhance the development of practical solutions to confront modern agricultural constraints. Here, a critical review of mutant collections constructed by various laboratories during the past decade is provided. We used Magnaporthe oryzae and Fusarium graminearum studies to show how mutant screens contribute to bridge existing knowledge gaps in pathogenicity and fungal–host interactions.     

Transgenic plants of Vitis vinifera cv. Seyval blanc

Leaf discs of grapevine cv. Seyval blanc originating from in vitro cultures were transformed with Agrobacterium tumefaciens strain LBA 4404 harbouring the vector pGJ42 carrying genes for chitinase and RIP (ribosome-inactivating protein) in an attempt to improve fungal resistance. The gene for neomycin phosphotransferase II (nptII) was used as the selectable marker gene. The explants were cocultivated for 2 days with recombinant Agrobacteria and then submitted to selection on NN69 medium containing 100 mg/l kanamycin. Successful regeneration and conversion of transgenic plantlets were obtained. Stable integration of foreign DNA was confirmed by PCR and Southern blot analyses, and protein expression was detected by Western blot. The regenerated transgenic plants were adapted to the greenhouse and showed no evidence of phenotypical alterations. The foreign genes introduced into the transformed plants did not effect the expected improvement in fungal disease resistance under field conditions for the major pests Uncinula necator and Plasmopara viticola.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Inheritance of Fusarium wilt resistance introgressed from Solanum aethiopicum Gilo and Aculeatum groups into cultivated eggplant (S. melongena) and development of associated PCR-based markers

The two eggplant relatives Solanum aethiopicum gr. Gilo and Solanum aethiopicum gr. Aculeatum (=Solanum integrifolium) carry resistance to the fungal wilt disease caused by Fusarium oxysporum f. sp. melongenae, a worldwide soil-borne disease of eggplant. To introgress the resistance trait into cultivated eggplant, the tetraploid somatic hybrids S. melongena + S. aethiopicum and S. melongena + S. integrifolium were used. An inheritance study of the resistance was performed on advanced anther culture-derived androgenetic backcross progenies from the two somatic hybrids. The segregation fitted a 3 resistant (R): 1 susceptible (S) ratio in the selfed populations and a 1R:1S ratio in the backcross progenies for the trait derived from S. aethiopicum and S. integrifolium. These ratios are consistent with a single gene, which we designated as Rfo-sa1, controlling the resistance to Fusarium oxysporum f. sp. melongenae. The allelic relationship between the resistance genes from S. aethiopicum and S. integrifolium indicate that these two genes are alleles of the same locus. Bulked Segregant Analysis (BSA) was performed with RAPD markers on the BC₃/BC₅ resistant advanced backcross progenies, and three RAPD markers associated with the resistance trait were identified. Cleaved Amplified Polymorphic Sequences (CAPSs) were subsequently obtained on the basis of the amplicon sequences. The evaluation of the efficiency of these markers in predicting the resistant phenotype in segregating progenies revealed that they represent useful tools for indirect selection of Fusarium resistance in eggplant.     

Ergosterol concentration and variability in genotype-by-pathogen interaction for grain mold resistance in sorghum

A lack of understanding of host-by-pathogen relations can hinder the success of breeding for resistance to a major disease. Fungal strain pathogenicity has to be understood from the virulence it can cause on susceptible genotypes and host resistance indicates which genotypes have resistance genes. Where the two worlds meet lies the place where researchers match the prevalent pathogen in the area of production with resistant varieties. This paper uses ergosterol concentration analysis as a measure of fungal biomass accumulation to assess levels of resistance in host genotypes. 11 sorghum genotypes were inoculated with 5 strains of fungi that are known to be associated with grain mold disease of sorghum. The resulting interaction was analyzed using GGE Biplot analysis and Cluster analysis which showed that none of the genotypes were resistant to Phoma sorghina and Curvularia lunata. Three genotypes were resistant to Fusarium thapsinum. One fungal strain (Alternaria alternata) does not contribute any significant damage in the grain mold disease. Fusarium graminearum causes very little grain mold disease. There was no correlation between the fungal strains. Visual scoring did not correlate with ergosterol accumulation. Resistance to grain mold in sorghum is shown to be due to vertical or specific resistance genes. Sorghum breeders should, therefore, identify predominant fungal strains in their localities and then locate and tag these resistance genes in their germplasm and pyramid them in commercial varieties.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity

Phospholipase C (PLC) generates various second messenger molecules and mediates phospholipid hydrolysis. In recent years, the important roles of plant and fungal PLC in disease resistance and pathogenicity, respectively, have been determined. However, the roles of PLC in plants and fungi are unintegrated and relevant literature is disorganized. This makes it difficult for researchers to implement PLC-based strategies to improve disease resistance in plants. In this comprehensive review, we summarize the structure, classification, and phylogeny of the PLCs involved in plant biotic stress resistance and fungal pathogenicity. PLCs can be divided into two groups, nonspecific PLC (NPC) and phosphatidylinositol-specific PLC (PI-PLC), which present marked differences in phylogenetic evolution. The products of PLC genes in fungi play significant roles in physiological activity and pathogenesis, whereas those encoded by plant PLC genes mediate the immune response to fungi. This review provides a perspective for the future control of plant fungal diseases.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> with thaumatin II gene to control fungal diseases

Transgenic plants of hyacinth (Hyacinthus orientalis L.) cvs. Edisson and Chine Pink have been obtained by Agrobacterium-mediated transformation. Leaf explants of the both hyacinth cultivars regenerated shoots on MS medium containing 2.2 μM BAP and 0.3 μM NAA at a frequency of 95%. A. tumefaciens strain CBE21 carrying binary vector pBIThau35 was used for transformation. Plasmid pBIThau35 has been produced by cloning preprothaumatin II cDNA into pBI121 instead of uidA gene. Inoculated leaf explants formed calli and shoots at high frequency on selective medium with 100 mg l⁻¹ kanamycin. Four hyacinth transgenic lines of cv. Chine Pink and one line of cv. Edisson have been selected on medium containing 200 mg l⁻¹ kanamycin. The insertion of thaumatin II gene into hyacinth genome has been confirmed by PCR-analysis. All transgenic plants expressed substantial amounts of thaumatin II (between 0.06 and 0.28% of the total soluble protein). Hyacinth transgenic lines were assayed for resistance to the pathogenic fungi Fusarium culmorum and Botrytis cinerea. There were no significant differences between nontransformed control and transgenic leaves of both cultivars. At the same time the bulbs of the transgenic line Н7401 cv. Chine Pink showed the higher level of resistance to B. cinerea, the bulbs of the transgenic line Н7404 were more resistant to F. culmorum. In both cases the signs of the fungal disease were developed more slowly. The resistance of the bulbs cv. Edisson line to these fungi was not changed. All transgenic hyacinth plant were successfully transferred to soil for further evaluation.     

Molecular mapping of wilt resistance genes in chickpea

Fusarium wilt is a widespread and serious chickpea disease caused by the soil-borne fungus Fusarium oxysporum f.sp. ciceri (Foc). We evaluated an F₉ recombinant inbred line population of chickpea for resistance to three Foc races (1, 2 and 3) in pot culture experiments and identified flanking and tightly linked DNA markers for the resistance genes. The simple sequence repeat markers H3A12 and TA110 flanked the Foc1 locus at 3.9 and 2.1 cM, respectively, while Foc2 was mapped 0.2 cM from TA96 and 2.7 cM from H3A12. The H1B06y and TA194 markers flanked the Foc3 locus at 0.2 and 0.7 cM, respectively. These markers were also validated using 16 diverse chickpea genotypes. Identification of tightly linked flanking markers for wilt resistance genes will be useful for their exploitation in breeding programs and to understand the mechanism of resistance and evolution of the genes. S. J. M. Gowda and P. Radhika contributed equally to this study.     

Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177

Summary This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.     

RFLP mapping of I1, a new locus in tomato conferring resistance against Fusarium oxysporum f. sp. lycopersici race 1

Summary The inheritance and linkage relationships of a gene for resistance to Fusarium oxysporum f. sp. lycopersici race 1 were analyzed. An interspecific hybrid between a resistant Lycopersicon pennellii and a susceptible L. esculentum was backcrossed to L. esculentum. The genotype of each backcross-1 (BC₁) plant with respect to its Fusarium response was determined by means of backcross-2 progeny tests. Resistance was controlled by a single dominant gene, I1, which was not allelic to I, the traditional gene for resistance against the same fungal pathogen that was derived from L. pimpinellifolium. Linkage analysis of 154 molecular markers that segregated in the BC₁ population placed I1 between the RFLP markers TG20 and TG128 on chromosome 7. The flanking markers were used to verify the assignment of the I1 genotype in the segregating population. The results are discussed with reference to the possibility of cloning Fusarium resistance genes in tomato.     

Characterization of a 20 kDa DNase elicitor from Fusarium solani f. sp. phaseoli and its expression at the onset of induced resistance in Pisum sativum

DNase released from Fusarium solani f. sp. phaseoli (Fsph DNase) has previously been reported to induce pathogenesis-related (PR) genes, phytoalexin accumulation and disease resistance against subsequent challenge with the true pea pathogen, Fusarium solani f. sp. pisi (Fspi). This report is a further analysis of DNase production with probes specific for both the gene and protein. N-terminal analysis of the ≈20 kDa Fsph DNase protein facilitated both the development of anti-Fsph DNase antiserum and the cloning of the Fsph DNase gene. Utilizing the anti-Fsph DNase antiserum to prepare an affinity column, we demonstrated that the retention and recovery of the DNase activity was associated with this protein. Fsph DNase protein was detectable by Western analysis in both the fungi and plant cytoplasm within 6–8 h following inoculation of the pea endocarp surface. Partially purified DNase detected via catalytic activity began accumulating within pea tissue at 3 h post-inoculation. Enhanced fragmentation of pea DNA occurred within 5 h following treatment of pods with Fsph DNase or inoculations with the two fungi. DNA cleavage within the nuclei of endocarp pea cells was detectable via a TUNEL assay at 3 h post-inoculation. As a result of these findings, we propose that the entrance of Fsph DNase into the pea cell and the signalling of plant defence responses is temporally associated with the damage of host DNA.     

Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.)

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis is one of the most devastating diseases in melon production worldwide. The most effective control measure available is the use of resistant varieties. Identifying molecular markers linked to resistance genes can serve as a valuable tool for the selection of resistant genotypes. Bulked segregant analysis was used to identify markers linked to the Fom-2 genes, which confers resistance to races 0 and 1 of the fungal pathogen. Pooled DNA from homozygous resistant or homozygous susceptible progeny of F₂ cross between MR-1 and AY was screened using 240 PstI/MseI and 200 EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified, all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cM, respectively. Moreover, AFLP marker AGG/CCC and the previously identified RAPD marker 596-1 cosegregated with Fom-2. These two dominant markers were converted to co-dominant markers by designing specific PCR primers that produced product length polymorphisms between the parents. A survey of 45 melon genotypes from diverse geographic origins with the co-dominant markers demonstrated a high correlation between fragment size and the resistance phenotype. These markers may therefore be useful in marker-assisted breeding programs.     

黄花倒水莲内生真菌生物活性评价及HNLF-44菌株鉴定

为充分开发黄花倒水莲(Polygala fallax)的内生真菌资源，获得具有抗植物病原真菌、抗氧化活性的内生真菌，该文以黄花倒水莲内生真菌为研究对象，使用平板对峙法检测内生真菌对6种植物病原真菌的抑菌活性，测定内生真菌发酵液的DPPH清除自由基能力和总还原能力，评价内生真菌的抗氧化活性，并对具有强抑菌活性和抗氧化活性的菌株进行形态和ITS鉴定。结果表明：(1)黄花倒水莲内生真菌中有2株内生真菌对香蕉专化尖孢镰刀菌、柑橘树脂病菌、叶点霉菌、香蕉具条叶斑病菌、茄病镰刀菌、三七根腐病菌具有明显的抑菌活性，抑菌率在50.3%～91.4%之间，其中HNLF-5对柑橘树脂病菌的抑菌率为73.2%,HNLF-44对香蕉专化尖孢镰刀菌抑菌率为91.4%。(2)内生真菌发酵液具有良好的抗氧化活性，DPPH清除率均在80%以上，总还原能力吸光值范围为0.279 2～0.748 8。(3)HNLF-44菌株为链格孢属真菌。该研究表明，药用植物黄花倒水莲内生真菌具有较好的生物活性，为后续从黄花倒水莲内生真菌中挖掘潜在新型抑菌活性和抗氧化活性物质奠定了基础。     

Molecular markers linked to the blast resistance gene <Emphasis Type="Italic">Pi-z</Emphasis> in rice for use in marker-assisted selection

Rice blast, caused by the fungal pathogen Pyricularia grisea, is a serious disease affecting rice-growing regions around the world. Current methods for identification of blast-resistant germplasm and progeny typically utilize phenotypic screening. However, phenotypic screens are influenced by environmental conditions and the presence of one resistance gene can sometimes phenotypically mask other genes conferring resistance to the same blast race. Pi-z is a dominant gene located on the short arm of chromosome 6 that confers complete resistance to five races of blast. Using sequence data found in public databases and degenerate primer pairs based on the P-loop, nucleotide binding sites and kinase domain motifs of previously cloned resistance genes, we have developed PCR-based DNA markers that cosegregate with the gene. These markers are polymorphic in a wide range of germplasm, including the narrow crosses characteristic of applied rice-breeding programs. They can now be used as a low cost, high-throughput alternative to conventional phenotypic screening for direct detection of blast resistance genes, allowing rapid introgression of genes into susceptible varieties as well as the incorporation of multiple genes into individual lines for more-durable blast resistance.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by D. Mackill     

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.     

Production of transgenic potato exhibiting enhanced resistance to fungal infections and herbicide applications

Potato (Solanum tuberosum L.), one of the most important food crops, is susceptible to a number of devastating fungal pathogens in addition to bacterial and other pathogens. Producing disease-resistant cultivars has been an effective and useful strategy to combat the attack of pathogens. Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus strain HUT 6037 and bialaphos resistance (bar) genes in a binary plasmid vector, pEKH1. Polymerase chain reaction (PCR) analysis revealed that the ChiC and bar genes are integrated into the genome of transgenic plants. Different insertion sites of the transgenes (one to six sites for ChiC and three to seven for bar) were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of the ChiC gene at the messenger RNA (mRNA) level was confirmed by Northern blot analysis and that of the bar gene by herbicide resistance assay. The results obviously confirmed that the ChiC and bar genes are successfully integrated and expressed into the genome, resulting in the production of bialaphos-resistant transgenic plants. Disease-resistance assay of the in vitro and greenhouse-grown transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight).     

Transposable elements in the fungal plant pathogenFusarium oxysporum

The genome of the fungal plant pathogenFusarium oxysporum contains at least six different families of transposable elements. Representatives of both DNA transposons and retrotransposons have been identified, either by cloning of dispersed repetitive sequences (Foret andpalm) or by trapping in the nitrate reductase gene (Fot1, Fot2 Impala andHop).Fot1 andImpala elements are related to theTc1 andmariner class of transposons. These transposable elements can affect gene structure and function in several ways: inactivation of the target gene through insertion, diversification of the nucleotide sequence by imprecise excisions, and probably chromosomal rearrangements as suggested by the extensive karyotype variation observed among field isolates. Comparisons of the distribution of these elements inFusarium populations have improved our understanding of population structure and epidemiology and provided support for horizontal genetic transfer. Also they could be developed as genetic tools for tagging genes, a cloning strategy that is particularly promising in imperfect fungi.     

Fungal endophytes from the three-leaved caper,Crataeva magna (Lour.) DC. (Capparidaceae)

Fungal endophytes were isolated from Crataeva magna, a medicinal plant growing along the streams and rivers, constituting riparian vegetation in Karnataka, southern India. Fresh bark and twig pieces were used for the isolation using standard methods. Ninety-six endophytic fungal isolates were isolated from 800 bark and twig segments. Mitosporic fungi represented as a major group (85%) followed by zygomycetes (10%) and ascomycetes (5%). Bark samples contained more endophytes than twig samples. Verticillium, Nigrospora oryzae and Fusarium verticilloides were the dominant fungal endophytes.     

Biological control of crown rot of bananas with Pichia anomala strain K and Candida oleophila strain O

The antagonistic activity of two yeast strains (Pichia anomala (E.C. Hansen) Kurtzman, strain K and Candida oleophila Montrocher, strain O) against the parasitic complex responsible for banana crown rot was evaluated. The strains were applied at three different concentrations (10⁶, 10⁷, 10⁸ cfu/ml) and their efficacy tested in vivo on three separate fungi (Colletotrichum musae (Berk. & Curt.) Arx, Fusarium moniliforme Sheldon, and Cephalosporium sp.) and on a parasitic complex formed by association of these three fungi. At the concentrations used C. musae appeared to be the most pathogenic. The complex showed intermediate aggressiveness between C. musae and both other fungi.Statistically significant antagonistic effects were observed on C. musae, F. moniliforme, and the fungal complex. The highest protection level (54.4%) was observed with strain O added at 10⁸ cfu/ml on crowns previously inoculated with the fungal complex. The level was lower when the fungi were inoculated separately.Furthermore, the antagonistic effect was strongly reinforced when strain O at 10⁸ cfu/ml was applied 24 h before fungal complex inoculation (59.9%), as compared to its application 15 min (24.3%) or 3 h (27.3%) after fungal complex inoculation. Bananas showed increased susceptibility to the fungal complex from March to June, and this influenced the level of protection by yeast, which decreased over the same period. A strict negative correlation (R² = 0.83) was highlighted between susceptibility of banana to crown rot and protection provided by yeast.