Monilinia species in Hungary: morphology, culture characteristics, and molecular analysis

Monilinia is a well-known pathogen of fruit trees affecting fruit production all over the world. Three species of the Monilinia genus are particularly important with regard to fruit trees and ornamentals, causing serious blossom and twig blight and brown rot in fruits: Monilinia fructicola, Monilinia fructigena, and Monilinia laxa. In this study, Monilinia isolates were compared and identified using classical and molecular methods. Morphological and culture characteristics were determined and pathogenicity testing performed. In addition, internal transcribed spacer regions and a genomic sequence with unknown function were analyzed and compared with sequence data from other Monilinia species in an international database. Four Monilinia/Monilia species were identified: M. fructicola, Monilia polystroma, M. fructigena, and M. laxa. M. fructicola was isolated from imported peach fruits. M. polystroma was first reported from Hungary and Europe on apple shoots and fruits. M. fructigena was identified on tea-rose hybrid pseudofruits, which is the first occurrence of this pathogen on this host. M. laxa causes brown rot of grapes, which has only been reported in New Zealand. Substitutions and insertions were detected when comparing M. laxa, M. fructigena, and M. polystroma sequences. In the genomic sequence with unknown function, three repetitive sequence motifs were identified in different numbers, depending on species and isolate. On the phylogram produced in this analysis, the Hungarian M. polystroma isolate (UFT) and M. polystroma reference isolates localized at a different branch than the closely related M. fructigena isolates and other Monilinia species.     

Postharvest biocontrol of Monilinia laxa,Monilinia fructicola and Monilinia fructigena on stone fruit by two Aureobasidium pullulans strains

The antagonistic effects of yeasts, L1 and L8, isolated from carposphere of ‘Redhaven’ peaches were tested for the first time in the same experiment against three Monilinia species (Monilinia laxa, Monilinia fructicola and Monilinia fructigena) in in vitro and in vivo trials. The two antagonists were selected after preliminary assays for their ability to reduce brown rot in peaches and nectarines, and both were identified by molecular and morphological tools as Aureobasidium pullulans. In in vivo trials, neither the autoclaved cells, nor the sterile culture filtrates of either antagonist showed any significant reduction of rot incidence produced by inocula of the three Monilinia species, while the washed cells of L1 and L8 completely inhibited M. laxa and M. fructicola rots and reduced M. fructigena infections by 70% and 90%, respectively. In other trials, nectarines treated with antagonist cells and inoculated with the pathogens were stored at 0 °C for 21 days, plus 7 days at 20 °C. The low temperature reduced brown rot development, since all fruit were free from disease symptoms on removal from cold storage. However after 7 d at 20 °C, untreated fruit were rotted over 45% depending on the Monilinia species but the antagonists completely inhibited M. laxa and M. fructicola, while M. fructigena infections were reduced by 89.8% and 91.2% by L1 and L8, respectively. For both strains, 10⁸ CFU ml^?1 was the most active concentration, although L1 showed good activity at a concentration of 10⁷ CFU ml^?1. Isolate L8 at the concentration of 10⁷ CFU ml^?1 was ineffective against M. fructicola and M. fructigena, showing no difference between treated fruit and the control, excepting the case of nectarines inoculated with M. laxa, where L8 at the concentration of 10⁷ CFU ml^?1 reduced the brown rot infections with respect to the control. The increase in population density of A. pullulans strains L1 and L8 in the wounds of nectarines stored at 0° or 20 °C was low but sufficient to control brown rot. In conclusion, the present preliminary study identified two antagonistic strains of A. pullulans as active ingredients for the development of biofungicides for postharvest application against three Monilinia species that are responsible for high economic losses in stone fruit crops.     

Genetic Diversity in Monilinia laxa Populations in Peach Orchards in Spain

One‐hundred and forty‐four random amplified polymorphic DNA markers, of which 59 were polymorphic and 85 monomorphic, were used to assess the genetic diversity and to study the structure of Monilinia laxa populations in Spain. Twenty‐one isolates collected from several orchards (subpopulations), in various years and in various hosts, were used. The analysis of population structure revealed that genetic diversity within orchards (H_S) accounted for 97% of the total genetic diversity (H_T), while genetic diversity among the orchards represented only 3%. The relative magnitude of gene differentiation between subpopulations (G_ST) and the estimate of the number of migrants per generation (N_m) averaged 0.032 and 15.1 respectively. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrogram was independent of whether they came from the same or different orchards. There was no relationship between clustering among isolates from distinct years and hosts. The relative importance of several evolutionary forces in populations of M. laxa is discussed, together with implications for the management of brown rot.     

Genetic diversity in Monilinia laxa populations in stone fruit species in Hungary

The objectives of this study were firstly, to determine the genetic diversity of Monilinia laxa isolates from Hungary, using the PCR-based inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) technique; secondly, to prepare genetic diversity groups based on the dendrograms; and finally, to select some relevant isolates to study their fungicide sensitivity. 55 and 77 random amplified polymorphic ISSR and RAPD markers, of which 23 and 18 were polymorphic and 32 and 59 monomorphic, respectively, were used to assess the genetic diversity and to study the structure of M. laxa populations in Hungary. 27 isolates out of 57 ones were confirmed as M. laxa from several orchards (subpopulations) in three geographical regions, in various inoculum sources and in various hosts, were used. 10 fungicides and 12 isolates selected from genetic diversity groups based on the ISSR dendrograms were used to determine the fungicide sensitivity of the selected isolates. The analysis of population structure revealed that genetic diversity within locations, inoculum sources and host (H _S ) accounted for 99 % of the total genetic diversity (H _T ), while genetic diversity among locations, inoculum sources and host represented only 1 %. The relative magnitude of gene differentiation between subpopulations (G _ST ) and the estimate of the number of migrants per generation (Nm) averaged 0.005–0.009 and 53.9–99.2, respectively, for both ISSR and RAPD data set. The results obtained in dendrograms were in accordance with the gene diversity analysis. Grouping of isolates in the dendrograms was irrespective of whether they came from the same or different geographical locations. There was no relationship between clustering among isolates from inoculum sources and hosts. In the fungicide sensitivity tests, five isolates out of 12 were partly insensitive to boscalid+piraclostrobin, cyprodinil, fenhexamid or prochloraz. Obtained results in genetic diversity of M. laxa populations are discussed together with implications for the management of brown rot.     

Overwintering of Monilinia fructicola in Stone Fruit Orchards in Northern China

Survival of Monilinia fructicola on the surface of mummified fruit and in peduncles and shoots of stone fruit trees infected by M. fructicola in the previous season was studied from 2003 to 2006 in orchards in the agricultural region of Beijing. Viable conidia of M. fructicola were consistently detected on fruit mummies from mid‐March to the end of April. During flowering (in mid‐April), studies in five peach orchards showed that 33–87% of mummified fruit bore viable conidia. The germination rate of conidia on diseased fruit was about 64% in autumn. It decreased to 24% in mid‐winter when the fruit was completely mummified, and in the following year to 2–4% in early spring. Viable M. fructicola was consistently detected in peach and nectarine shoots collected in winter and spring. In general, viable M. fructicola in peduncles was detected from mummified fruit of 11–27% branches and from asymptomatic plant tissues of 3–20% branches. Sporulation of M. fructicola was observed on peduncles in seven of eight surveys, and the percentage of branches containing viable M. fructicola in peduncles in contact with mummified fruits ranged from 18% to 40%. This study demonstrates that the tree‐borne mummified fruit and the peduncles could be the parts of trees where M. fructicola can survive the winter in orchards in suburban Beijing.     

High‐Throughput Assessment and Genetic Investigation of Vegetative Compatibility in Verticillium dahliae

Classification of isolates into vegetative compatibility groups (VCGs) using nitrate‐non‐utilizing (nit) mutants has been widely used for the characterization of Verticillium dahliae populations. However, certain methodological limitations prevent its application on a large scale. Furthermore, systematic investigations into the genetics underlying complementation tests between nit mutants of fungal isolates (i.e. heterokaryon formation) are lacking for Verticillium species. In this work, a diverse collection of 27 V. dahliae isolates – including representatives of all VCGs, both mating types, and heterokaryon self‐incompatible isolates – was employed for the development and optimization of (i) a protocol for the rapid generation of nit mutants of V. dahliae isolates using UV‐irradiation and (ii) a reproducible high‐throughput procedure for complementation tests between nit mutants in liquid cultures using 96‐well microplates. The genetic analysis of selected heterokaryons demonstrated that the frequently encountered ‘weak’ cross‐reactions between VCGs and their subgroups can be actually heterokaryotic, implying the absence of strict genetic barriers between VCGs. In conclusion, we provide in this work an optimized method for the high‐throughput VCG assignment of V. dahliae populations and a genetic analysis of heterokaryons that may have serious implications for the interpretation of VCG classification data. These advancements in the available methodology and the genetic background of vegetative compatibility grouping may contribute to a better understanding of the population biology of V. dahliae and possibly other mitosporic fungi.     

Vegetative Compatibility Among Isolates of Colletotrichum gloeosporioides from Yam (Dioscorea spp.) in Nigeria

Isolates of Colletotrichum gloeosporioides obtained from yam‐based cropping systems in Nigeria, previously characterized on the basis of morphology, virulence and rDNA internal transcribed spacer (ITS) sequence variation were further compared for vegetative compatibility (VC). Chlorate‐resistant nitrate non‐utilizing (nit) mutants were generated from the isolates and used in complementation (heterokaryon) tests. Tests of VC between complementary mutants from different isolates indicated the presence of several genotypes within a single field, suggesting limited clonal spread. In some cases, isolates obtained from the same lesion were observed to belong to different vegetative compatibility groups (VCGs). No compatibility was observed between isolates of the highly virulent slow‐growing grey (SGG), the moderately virulent fast‐growing salmon (FGS) and the avirulent/weakly virulent fast‐growing grey (FGG) strains. Forty‐one C. gloeosporioides isolates belonged to 28 VCGs, giving a genotype diversity estimate of 0.68. This diversity confirmed the high variability of the pathogen population as revealed by previous characterization studies, however, a correlation between VCGs and isolate groupings based on morphology and virulence was not found. The finding that an isolate from weed was compatible with yam isolates indicated that transfer of important traits, such as virulence, may take place between isolates from yam and non‐yam hosts. The VCG diversity revealed by this study suggests that in addition to asexual reproduction, sexual reproduction may play an important role in the epidemiology of anthracnose on yam.     

Environmental distribution and genetic diversity of vegetative compatibility groups determine biocontrol strategies to mitigate aflatoxin contamination of maize by Aspergillus flavus

Maize infected by aflatoxin‐producing Aspergillus flavus may become contaminated with aflatoxins, and as a result, threaten human health, food security and farmers' income in developing countries where maize is a staple. Environmental distribution and genetic diversity of A. flavus can influence the effectiveness of atoxigenic isolates in mitigating aflatoxin contamination. However, such information has not been used to facilitate selection and deployment of atoxigenic isolates. A total of 35 isolates of A. flavus isolated from maize samples collected from three agro‐ecological zones of Nigeria were used in this study. Ecophysiological characteristics, distribution and genetic diversity of the isolates were determined to identify vegetative compatibility groups (VCGs). The generated data were used to inform selection and deployment of native atoxigenic isolates to mitigate aflatoxin contamination in maize. In co‐inoculation with toxigenic isolates, atoxigenic isolates reduced aflatoxin contamination in grain by > 96%. A total of 25 VCGs were inferred from the collected isolates based on complementation tests involving nitrate non‐utilizing (nit^?) mutants. To determine genetic diversity and distribution of VCGs across agro‐ecological zones, 832 nit^? mutants from 52 locations in 11 administrative districts were paired with one self‐complementary nitrate auxotroph tester‐pair for each VCG. Atoxigenic VCGs accounted for 81.1% of the 153 positive complementations recorded. Genetic diversity of VCGs was highest in the derived savannah agro‐ecological zone (H = 2.61) compared with the southern Guinea savannah (H = 1.90) and northern Guinea savannah (H = 0.94) zones. Genetic richness (H = 2.60) and evenness (E₅ = 0.96) of VCGs were high across all agro‐ecological zones. Ten VCGs (40%) had members restricted to the original location of isolation, whereas 15 VCGs (60%) had members located between the original source of isolation and a distance > 400 km away. The present study identified widely distributed VCGs in Nigeria such as AV0222, AV3279, AV3304 and AV16127, whose atoxigenic members can be deployed for a region‐wide biocontrol of toxigenic isolates to reduce aflatoxin contamination in maize.     

Identification and Characterization of Benzimidazole Resistance in Monilinia fructicola from Stone Fruit Orchards in California

Low and high levels of resistance to the benzimidazole fungicides benomyl and thiophanate-methyl were observed in field isolates of Monilinia fructicola, which is the causative agent of brown rot of stone fruit. Isolates that had low levels of resistance (hereafter referred to as LR isolates) and high levels of resistance (hereafter referred to as HR isolates) were also cold and heat sensitive, respectively. Results from microsatellite DNA fingerprints showed that genetic identities among the populations of sensitive (S), LR, and HR isolates were very high (>0.96). Analysis of DNA sequences of the β-tubulin gene showed that the LR isolates had a point mutation at codon 6, causing a replacement of the amino acid histidine by tyrosine. Codon 198, which encodes a glutamic acid in S and LR isolates, was converted to a codon for alanine in HR isolates. Based on these point mutations in the β-tubulin gene, allele-specific PCR assays were developed for rapid detection of benzimidazole-resistant isolates of M. fructicola from stone fruit.     

Genetic Diversity of Populations of Monilinia fructicola (Fungi,Ascomycota, Helotiales) from China

ABSTRACT. The genetic variation among 128 isolates of Monilinia fructicola (Fungi, Ascomycota, Helotiales) from China was analyzed using Inter‐Simple Sequence Repeat (ISSR) markers and compared with those of samples from California, USA and New Zealand. A total of 72 reproducible DNA fragments were scored, of which 87.5% (63/72) were polymorphic. The Nei's gene diversity and Shannon's diversity indices of three Chinese regional populations were very similar to that from California. However, several differences were observed among geographic populations of M. fructicola from both within China and between China and California. The analysis of molecular variance (AMOVA) of isolates from different geographic locations suggested that most of the observed genetic variation was found within populations. Results of this study are inconsistent with the hypothesis that the Chinese populations of M. fructicola were derived from a single or few recent migrants from other countries. Instead, our results suggest that M. fructicola has been in China long before its first official recording in 2003.     

Nitrate-nonutilizing mutants ofGibberella zeae (Fusarium graminearum) and their use in determining vegetative compatibility

Twenty-four single-spore isolates ofFusarium graminearum were obtained from scabby wheat seeds or glumes collected from 23 locations in Kansas in 1990. All isolates were sexually fertile and homothallic. Nitrate-nonutilizing (nit) mutants of each isolate were generated on a medium amended with 1.5% KCIO₃. Of 378 mutants, 161 were able to utilize nitrite and hypoxanthine (nit1), 165 utilized hypoxanthine but not nitrite (nit3), 47 utilized nitrite but not hypoxanthine (NitM), and 5 appeared to be global nitrogen regulatory mutants similar to the previously describednnu mutant. Complementation was tested by pairingnit1 mutants of each isolate with either a NitM or anit3 mutant from each isolate on media containing nitrate as the sole nitrogen source. Complementation was more pronounced whennit1 and NitM mutants were paired. Mutants were only able to complement with other mutants from the same wild-type isolate. Therefore, each wild-type isolate belonged to a genetically distinct vegetative compatibility group. The genetic diversity suggests that sexual genetic recombination may be important in the field.     

Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

Vegetative Compatibility,Pathogenicity and Virulence Diversity of Fusarium oxysporum f. sp. melongenae Recovered from Eggplant

Fusarium oxysporum (Schlechtend.: Fr.) f. sp. melongenae (Fomg) recovered from symptomatic eggplants from five eggplant‐growing areas in Turkey, including the south, west, north‐west, north and south‐east regions. The objective of this study was to investigate the genetic diversity of the Fomg isolates from different geographical location by pathogenicity and VCG tests. Three hundred and seventy‐four Fomg isolates were classified as highly virulent, virulent, moderately virulent and low virulent through pathogenicity assays. No correlation was observed between virulence of Fomg isolates and their locations. The nitrate non‐utilizing mutants (nit) were generated as nit1, nit3 and NitM, based on phenotyping of Fomg growth characteristics of the Fomg isolates on diagnostic media with various sources of nitrogen. The majority of nit mutants (39.4%) recovered were nit1 from minimal medium (MM) containing of 2.0% potassium chlorate (MMC). The most of Fomg isolates were identified as heterokaryon self‐compatible (HSC) based on their ability to form a stable heterokaryon, while four isolates were classified as heterokaryon self‐incompatible (HSI). A large amount of Fomg isolates were vegetatively compatible and assigned as members of the same VCG, whereas nit mutants of 10 Fomg isolates that did not complement with tester strains only paired by themselves (HSC), these isolates were termed vegetative incompatible (vic). The complementation of 33 isolates with tester strains was slow and quite weak, but not paired with themselves even though they are HSC. About 96.3% of the Fomg isolates were assigned to VCG 0320, while the remaining 3.7% were classified as vegetative incompatible group.     

Nutritional requirements of antagonists to peach twig blight,Monilinia laxa,in relation to biocontrol

Different carbon and nitrogen sources and accessory substances were tested to determine their effect on the growth and sporulation of the peach twig blight pathogen,Monilinia laxa, and of three of its antagonists (Penicillium frequentans, Penicillium purpurogenum andEpicoccum nigrum), since the success in twig blight biological control by treatments with the fungal antagonists depends on the type of nutrients added to the antagonist formulation. Combinations of sucrose-ammonium tartrate, glucose-(NH₄)₃PO₄-folic acid and lactose-KNO₃ were selected from these laboratory experiments because they enhanced the growth and sporulation ofP. frequentans, P. purpurogenum andE. nigrum, respectively, but notM. laxa. In glasshouse experiments, twig blight was reduced following the application of mixtures of antagonists with the corresponding enhancing nutrients.     

Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit

Biocontrol potential of Bacillus subtilis strain CPA-8 was tested against the main postharvest diseases of orange, apple and stone fruit. Previously, CPA-8 growth was characterized and its antifungal activity in vitro determined against Botrytis cinerea, Monilinia laxa, M. fructicola, Penicillium digitatum, P. expansum, and P. italicum. In vivo activity against these pathogens was tested by treating fruits with cells, endospores or cell free supernatants. CPA-8 treatments cannot control decay caused by P. digitatum and P. italicum on oranges. The higher concentrations of CPA-8 studied were effective in controlling B. cinerea on apple, showing grey mold incidence from 70 to 12.5% in comparison with 100% in the control. However, in general, CPA-8 treatments were not effective in controlling P. expansum. The best results of CPA-8 treatments were obtained in stone fruit against M. laxa and M. fructicola where most treatments resulted in brown rot incidence of 0% compared with 70 and 90% in the control. Based on these results, cultures, cells and cell free supernatants at different concentrations were tested against M. laxa and M. fructicola on stone fruit. Most bacterial concentrations were effective in controlling M. laxa and M. fructicola as well as or better than Serenade® Max, in some treatments showing brown rot incidences of 0% in comparison with 100% of control. Bacterial populations of CPA-8 were maintained stable or increased up to 2-log inside wounds, showing the ability of the bacteria to colonize injured tissues. Experimental evidence suggests that B. subtilis CPA-8 has biocontrol potential for control of postharvest disease on several fruit types.     

Potential diversity in vegetative compatibility groupings in the California population of Gibberella circinata

Pitch canker, caused by Gibberella circinata, is a disease affecting pines throughout the world. Although the pathogen is capable of sexual reproduction, natural populations are often comprised of very few vegetative compatibility groups (VCGs), which implies a predominance of asexual reproduction. However, even where outcrossing occurs, a population could have limited VCG diversity due to a low level of polymorphism at the loci governing vegetative compatibility (= vic loci). To determine whether this was the case for the California population of G. circinata, inter-fertile strains were crossed under laboratory conditions. Progeny of this cross included a minimum of 29 VCGs, which was consistent with segregation of vic alleles at five loci. Only two of these VCGs were known to occur naturally in California. Three VCGs accounted for 32.4 % of the progeny but 0 % of the California population. Overall, the results support the conclusion that outcrossing has been rare or absent in the California population of G. circinata.     

The Cytochrome P450 Lanosterol 14α-Demethylase Gene Is a Demethylation Inhibitor Fungicide Resistance Determinant in Monilinia fructicola Field Isolates from Georgia

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14α-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 μg of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position −117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.     

Genetic Diversity of Fusarium oxysporum Strains from Common Bean Fields in Spain

Fusarium wilt is an endemic disease in El Barco de Avila (Castilla y León, west-central Spain), where high-quality common bean cultivars have been cultured for the last century. We used intergenic spacer (IGS) region polymorphism of ribosomal DNA, electrophoretic karyotype patterns, and vegetative compatibility and pathogenicity analyses to assess the genetic diversity within Fusarium oxysporum isolates recovered from common bean plants growing in fields around El Barco de Avila. Ninety-six vegetative compatibility groups (VCGs) were found among 128 isolates analyzed; most of these VCGs contained only a single isolate. The strains belonging to pathogenic VCGs and the most abundant nonpathogenic VCGs were further examined for polymorphisms in the IGS region and electrophoretic karyotype patterns. Isolates belonging to the same VCG exhibited the same IGS haplotype and very similar electrophoretic karyotype patterns. These findings are consistent with the hypothesis that VCGs represent clonal lineages that rarely, if ever, reproduce sexually. The F. oxysporum f. sp. phaseoli strains recovered had the same IGS haplotype and similar electrophoretic karyotype patterns, different from those found for F. oxysporum f. sp. phaseoli from the Americas, and were assigned to three new VCGs (VCGs 0166, 0167, and 0168). Based on our results, we do not consider the strains belonging to F. oxysporum f. sp. phaseoli to be a monophyletic group within F. oxysporum, as there is no correlation between pathogenicity and VCG, IGS restriction fragment length polymorphism, or electrophoretic karyotype.     

Genetic relationships between virulence,vegetative compatibility and ISSR marker of Verticillium dahliae isolated from cotton

Verticillium dahliae is one of the most important pathogens causing Verticillium wilt. We studied the characterisation of the genetic relationship between virulence, vegetative compatibility groups (VCGs) and inter-simple sequence repeat (ISSR). A total of 48 V. dahliae isolates, in which 16 are collected from different cotton growing regions in China and 4 isolates belonged to all known VCGs, are used. Half of them were found highly virulent. Mutants (565) were obtained using the nitrate non-utilising mutant. These mutants were grouped into three VCGs: VCG1 (27 isolates), VCG 2 (14 isolates) and VCG 3 (7 isolates). Use of ISSR indicated two main clusters that were related to VCG and virulence. Genetic diversity lineages were obviously correlated to VCGs and ISSRs according to their geographical origin, virulence and ISSR genetic variation. This study could be useful to design and develop effective management strategies beside for quarantine purposes on Verticillium wilt control.     

MfOfd1 is crucial for stress responses and virulence in the peach brown rot fungus Monilinia fructicola

Monilinia fructicola is the most widely distributed species among the Monilinia genus in the world, and causes blossom blight, twig canker, and fruit rot on Rosaceae fruits. To date, studies on genomics and pathogenicity are limited in M. fructicola. In this study, we identified a redox-related gene, MfOfd1, which was significantly up-regulated at 1 hr after inoculation of M. fructicola on peach fruits. We used the clustered regulatory inter-spaced short palindromic repeats (CRISPR)/Cas9 system combined with homologous recombination to determine the function of the MfOfd1 gene. The results showed that the sporulation of knockdown transformants was reduced by 53% to 83%. The knockdown transformants showed increased sensitivity to H₂O₂ and decreased virulence on peach fruits compared to the wild-type isolate Bmpc7. It was found that H₂O₂ could stimulate the expression of MfOfd1 in the wild-type isolate. The transformants were also more sensitive to exogenous osmotic stress, such as glycerol, d -sorbitol, and NaCl, and to dicarboximide fungicides (iprodione and dimethachlon). These results indicate that the MfOfd1 gene plays an important role in M. fructicola in sporulation, oxidative response, osmotic stress tolerance, and virulence.