A new pathogenesis-related protein,LrPR4, from <Emphasis Type="Italic">Lycoris radiata</Emphasis>, and its antifungal activity against <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A new Lycoris radiata pathogenesis-related (PR)-4 gene, LrPR4 was isolated. LrPR4 encodes a 142 amino acid protein with a predicted molecular mass of 15.43 kDa and pI of 7.56. The putative LrPR4 shows high similarity to PR4 type proteins from various plant species and belongs to the Barwin family. Like other PR4s from monocot plants, LrPR4 protein contains a conserved Barwin domain and has a signal peptide at its N-terminus. The recombinant LrPR4 protein expressed in Escherichia coli showed activity towards hydrolysing RNA from L. radiata bulbs and antifungal activity. The results of this study suggest that LrPR4 may play a role in the disease resistance responses of plant against pathogen attacks though its antifungal activity.     

Virulence and Vegetative Compatibility of Dutch and Italian Isolates of Fusarium oxysporum f. sp. lilii

The virulence and vegetative compatibility of eight Dutch and four Italian isolates of Fusarium oxysporum obtained from lily were compared. The virulence was tested by determination of the specific interaction between the Fusarium isolates and eight lily cultivars. A specific interaction was not found, so the existence of races was not demonstrated. Six of the twelve isolates turned out to be non-pathogenic for lily. The pathogenic isolates fell in four vegetative compatibility groups. No vegetative compatibility was found between isolates of F. oxysporum f. sp. lilii and those of f. sp. gladioli.     

Evolutionary Relationships among the Fusarium oxysporum f. sp. cubense Vegetative Compatibility Groups

Fusarium oxysporum f. sp. cubense, the causal agent of fusarium wilt of banana (Musa spp.), is one of the most destructive strains of the vascular wilt fungus F. oxysporum. Genetic relatedness among and within vegetative compatibility groups (VCGs) of F. oxysporum f. sp. cubense was studied by sequencing two nuclear and two mitochondrial DNA regions in a collection of 70 F. oxysporum isolates that include representatives of 20 VCGs of F. oxysporum f. sp. cubense, other formae speciales, and nonpathogens. To determine the ability of F. oxysporum f. sp. cubense to sexually recombine, crosses were made between isolates of opposite mating types. Phylogenetic analysis separated the F. oxysporum isolates into two clades and eight lineages. Phylogenetic relationships between F. oxysporum f. sp. cubense and other formae speciales of F. oxysporum and the relationships among VCGs and races of F. oxysporum f. sp. cubense clearly showed that F. oxysporum f. sp. cubense''s ability to cause disease on banana has emerged multiple times, independently, and that the ability to cause disease to a specific banana cultivar is also a polyphyletic trait. These analyses further suggest that both coevolution with the host and horizontal gene transfer may have played important roles in the evolutionary history of the pathogen. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system should sexual reproduction occur. Although, no sexual structures were observed, some lineages of F. oxysporum f. sp. cubense harbored MAT-1 and MAT-2 isolates, suggesting a potential that these lineages have a sexual origin that might be more recent than initially anticipated.Fusarium oxysporum Schlechtendahl emend. Snyder and Hansen is a cosmopolitan species (9) comprised of both pathogenic and nonpathogenic isolates (20). The pathogenic isolates of F. oxysporum cause fusarium wilt of several agricultural crops, and are accordingly subdivided into formae speciales (3, 26, 55). One of the economically more important and destructive formae speciales is the causal agent of fusarium wilt (Panama disease) of banana (Musa spp.), F. oxysporum f. sp. cubense (E. F. Smith) Snyder et Hansen. This disease has been reported in all banana production regions of the world, except those bordering the Mediterranean, Melanesia, Somalia, and some islands in the South Pacific (66, 77).A range of approaches are typically employed for the characterization of F. oxysporum f. sp. cubense isolates. Based on virulence to specific banana cultivars (66, 67), the pathogen may be classified into one of three races (i.e., races 1, 2, and 4), although this designation may be contingent on environmental conditions. For instance, genetically identical isolates of F. oxysporum f. sp. cubense are classified as race 4 isolates in the subtropics and as race 1 isolates in the tropics because they cause disease to Cavendish bananas under subtropical conditions only (67, 86). Based on vegetative compatibility, F. oxysporum f. sp. cubense isolates have been separated into 24 so-called vegetative compatibility groups (VCGs) (5, 29, 47, 68). Finally, various DNA-based tools have been used to separate F. oxysporum f. sp. cubense into a number of clonal lineages that more or less correspond to their grouping based on VCGs (6, 22, 38, 59).The evolutionary history of F. oxysporum f. sp. cubense is complex. Based on the results of phylogenetic studies (4-7, 22, 38, 57, 59). F. oxysporum f. sp. cubense represent multiple unrelated lineages, some of which are more closely related to other formae speciales of F. oxysporum than to other F. oxysporum f. sp. cubense lineages (3, 57, 59). This has lead to speculations that new pathogenic forms of F. oxysporum may be derived from other pathogenic and nonpathogenic members of this species (21). Factors such as coevolution with the plant host and the spread of virulence determinants via processes such as parasexuality, heterokaryosis, and sexual recombination also have been implicated in the evolution of this pathogen (11, 36, 37, 39, 64, 65, 69). Although parasexuality and heterokaryosis are known to occur in F. oxysporum (11, 39), sexual fruiting structures have never been observed in the species and only indirect evidence for sexual recombination has been detected (82). Indeed, the organization of the F. oxysporum f. sp. cubense mating type locus (MAT) is similar to those found in the closely related Gibberella fujikuroi (Sawada) Ito in Ito et K. Kimura complex and other heterothallic ascomycetes (2, 90).Development of appropriate disease management strategies and the selection of F. oxysporum f. sp. cubense-resistant banana cultivars may benefit from a better understanding of the diversity and evolutionary history of the pathogen. Although most previous DNA-based studies provided knowledge regarding the diversity of F. oxysporum f. sp. cubense, the genetic relatedness among the lineages identified in these studies remains uncertain (22). It is also not clear how the different races and VCGs of F. oxysporum f. sp. cubense are related to one another and to other isolates of F. oxysporum. Therefore, the main objective of this study was to resolve the relationships among the F. oxysporum f. sp. cubense VCGs and determine their relationships with other formae speciales and nonpathogenic members of F. oxysporum by using a multigene phylogenetic approach (8, 32, 52, 53, 62, 75, 91). To facilitate the rapid differentiation of the various F. oxysporum f. sp. cubense lineages, we also aimed to develop a diagnostic PCR-restriction fragment length polymorphism (RFLP) procedure. To evaluate the potential of F. oxysporum f. sp. cubense to reproduce sexually, sexual crosses among isolates of opposite mating types were attempted after PCR-based detection of the MAT-1 and MAT-2 idiomorphs (34).     

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K_s, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K_s, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K_s, about 30 mM).     

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.?oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events.     

Rearrangement of leaf traits with changing source-sink relationship in blueberry (Vaccinium corymbosum L.) leaves

The source-sink relationship is one of major determinants of plant performance. The influence of reproductive sink demand on light-saturated photosynthesis (P_max), dark respiration (R_D), stomatal conductance (g_s), intrinsic water-use efficiency (WUE_i), contents of soluble sugar (SSC), nitrogen, carbon, and photosynthetic pigments was examined in blueberry (Vaccinium corymbosum L. cv. ‘Brigitta’) during the final stage of rapid fruit growth. Measurements were performed three times per day on developed, sun-exposed leaves of girdled shoots with 0.1, 1, and 10 fruit per leaf (0.1F:L, 1F:L, and 10F:L, respectively) and nongirdled shoots bearing one fruit per leaf (NG). Girdling and lower fruit amount induced lower P_max, g_s, N, and total chlorophyll (Chl) and higher WUE_i, SSC, R_D, Chl a/b ratio and carotenoids-to-chlorophylls ratio (Car/Chl) for the 1F:L and 0.1F:L treatments. The impact of girdling was counterbalanced by 10F:L, with NG and 10F:L having similar values. Variables other than P_max, R_D, g_s, WUE_i, and SSC were unaffected throughout the course of the day. P_max and g_s decreased during the course of the day, but g_s decreased more than P_max in the afternoon, while WUE_i was increasing in almost all treatments. SSC increased from the morning until afternoon, whereas R_D peaked at noon regardless of the treatment. Generally, P_max was closely and negatively correlated to SSC, indicating that sugar-sensing mechanisms played an important role in regulation of blueberry leaf photosynthesis. With respect to treatments, P_max and N content were positively related, while R_D was not associated to substrate availability. The enhanced Car/Chl ratio showed a higher photoprotection under the lower sink demand. Changes in the source-sink relationship in ‘Brigitta’ blueberry led to a rearrangement of physiological and structural leaf traits which allowed adjusting the daily balance between carbon assimilation and absorbed light energy.     

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.     

镰刀菌诱导的泸定百合SSH文库构建及抗病相关基因筛选

该研究以泸定百合(Lilium sargentiae Wilson)为材料,构建其组培苗经百合尖孢镰刀菌侵染后的叶片SSH文库,从中筛选镰刀菌枯萎病抗病相关基因。从正向SSH文库中随机挑取300个单克隆测序后得到280条ESTs,进行功能比对分析后,除去未知功能、沉冗蛋白以及无同源序列,得到有功能的ESTs共168条,其功能涉及信号传导、蛋白质合成与代谢、抗病与防御、物质与能量代谢、转录相关等多种途径,其中有31条ESTs与抗病防御相关。从抗病防御相关基因中选取8条ESTs:过氧化氢酶、ATP结合盒转运蛋白(ABC transporter)、Kunitz型胰蛋白酶抑制剂4(Kunitz trypsin inhibitor 4)、丝氨酸乙醛酸氨基转移酶、多聚泛素、脂氧合酶I(Lipoxygenase I)、丝氨酸/苏安酸蛋白激酶(Serine/Threonine-protein kinase)、抗坏血酸过氧化物酶(Arabidopsis thaliana),通过RTPCR对其表达情况进行分析,发现经镰刀菌诱导后均为上调表达,推测它们可能参与了泸定百合镰刀菌枯萎病的抗病反应途径。     

Specific PCR-based marker for detection of pathogenic groups of Fusarium oxysporum f. sp. cucumerinum in India

For the detection of Fusarium oxysporum f. sp. cucumerinum pathogenic groups, a specific PCR-based marker was developed. Specific random amplified polymorphic DNA (RAPD) markers which identified in four pathogenic groups I, II, III, and IV were cloned into P^Gem-Teasy vector. Cloned fragments were sequenced, and used for developing sequence characterized amplified regions (SCAR) primers for detection of pathogenic groups. F. oxysporum f. sp. cucumerinum isolates belonging to four pathogenic groups in India, cucumber nonpathogenic F. oxysporum, F. oxysporum f. sp. moniliforme and melonis, Fusarium udum, and isolate of Alternaria sp. were tested using developed specific primers. A single 1.320 kb, 770 bp, 1.119 kb, and 771 bp fragment were amplified from pathogenic group I, II, III, and IV isolates, respectively. Results showed the PCR based marker, which used in this research work, could detect up to 1 ng of fungal genomic DNA. The specific SCAR primers and PCR technique developed in this research easily detect and differentiate isolates of each F. oxysporum f. sp. cucumerinum pathogenic groups.     

RAPD Characterization of Fusarium oxysporum Isolates Pathogenic on Argyranthemum frutescens L.

The random amplified polymorphic DNA (RAPD) technique was used to analyse total genomic DNA of 10 isolates of a new Fusarium oxysporum pathogenic on Argyranthemum frutescens (Paris daisy), by comparing them with representatives of the formae speciales basilici, chrysanthemi, cyclaminis, dianthi, gladioli, lilii, lycopersici, melonis, pisi, radicis‐lycopersici, tracheiphilum, and a non‐pathogenic isolate of F. oxysporum. A close genetic relatedness was observed among most of the new isolates from A. frutescens. These isolates also shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. A single isolate among those tested from diseased A. frutescens was placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. All the new isolates from A. frutescens, with the exception of the single divergent one, could be identified by their characteristic amplification profile, using selected random primers. A rapid protocol for DNA extraction directly from fungal colonies grown on Fusarium selective medium allowed the complete analysis in less than 4 h.     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.     

Naphthoquinones produced by Fusarium oxysporum isolated from citrus

Six naphthoquinone pigments are described which were produced by Fusarium oxysporum isolates obtained from roots of diseased citrus trees. These were 8-O-methylbostrycoidin, 9-O-methylfusarubin, 9-O-methylanhydro-fusarubin, 5-O-methyljavanicin, 5-O-methylsolaniol and 1,4-naphthalenedione-3,8-dihydroxy-5,7-dimethoxy-2-(2-oxopropyl). These naphthoquinones had not been previously identified from F. oxysporum isolates.     

Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

Production of zearalenone,nivalenol, moniliformin,and wortmannin from toxigenic cultures ofFusarium obtained from pasture soil samples collected in New Zealand

One culture ofF avenaceum, 4 cultures ofF oxysporum, and 11 cultures of Fsambucinum were isolated from soil samples of pasture in New Zealand in 1987. All cultures, when grown on rice media and fed to rats caused a weight loss in rats as well as toxic signs including hemorrhaging and congestion, uterine enlargement, and hematuria. 6 out of 16 cultures caused death in rat feeding tests.F oxysporum #1 killed rats (feeding test) within 5-12hrs. 10 cultures produced zearalenone (19 to 8,849 ppm), 8 cultures produced nivalenol (32 to 117 ppm), 1 culture,F sambucinum #8, produced wortmannin (40 ppm), and 5 cultures produced moniliformin (19 to 9,000ppm). We report for the first time the co-occurrence of zearalenone, nivalenol, and moniliformin produced byF sambucinum #3 in culture.F avenaceum #1 andF oxysporum cultures (nos 1, 2, and 3) produced moniliformin alone.F oxysporum #4 produced zearalenone alone as well.F sambucinum #5 caused erythema in the small intestine of rats and 100% mortality and did not produce any known toxin(s). Nivalenol when administered to the stomach of rats orally at levels 10, 20, and 40mg/kg body weight caused inflammation in the intestines, coma, and death. The mycotoxins T-2 toxin, HT-2 toxin, T-2 tetraol, diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyl-and 15-acetyldeoxynivalenol, depoxynivalenol, fusarenon-X, alpha-and beta-zearalenone, and fusarochromanone (TDP-1) were not detected in the extracts of these cultures.     

Genealogical concordance phylogenetic species recognition in the Fusarium oxysporum species complex

Fusarium oxysporum is an important plant and human pathogenic ascomycetous group, with near ubiquity in agricultural and non-cultivated ecosystems. Phylogenetic evidence suggests that F. oxysporum is a complex of multiple morphologically cryptic species. Species boundaries and limits of genetic exchange within this complex are poorly defined, largely due to the absence of a sexual state and the paucity of morphological characters. This study determined species boundaries within the F. oxysporum species complex using Genealogical Concordance Phylogenetic Species Recognition (GCPSR) with eight protein coding loci. GCPSR criteria were used firstly to identify independent evolutionary lineages (IEL), which were subsequently collapsed into phylogenetic species. Seventeen IELs were initially identified resulting in the recognition of two phylogenetic species. Further evidence supporting this delineation is discussed.     

Production of pectolytic and cellulolytic enzymes byfusarium oxysporum andF. moniliforme under different cultivation conditions

Six-day incubation was most suitable for production of pectolytic and cellulolytic enzymes byFusarium on different culture media. Czapek’s medium favoured maximum production of polygalacturonase (PG) and cellulase (C_x), peptone dextrose gave highest yields of pectin methyl galacturonase (PMG) withF. oxysporum. Cole’s medium was found to be poor for the enzyme production by both organisms. A positive correlation was observed between the growth rate of the pathogenic forms and their enzyme production. InF. oxysporum the PG secretion was maximum at pH 4.5 and inF. moniliforme at pH 5.0. PMG production optimum was at pH 5.5. No PG and PMG were produced above pH 7. InF. oxysporum the C_x activity was highest at pH 5.5 and inF. moniliforme at pH 4.5. Maximum PG and PMG activities were recorded at 35 °C in both pathogens. The C_x activity of both organisms was maximum at 45 °C but some carboxymethyl cellulose hydrolysis was found even at 60 °C.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.