Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays

ABSTRACT Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.     

PCR assays for the sugarcane rust pathogens Puccinia kuehnii and P. melanocephala and detection of a SNP associated with geographical distribution in P. kuehnii

Puccinia kuehnii and P. melanocephala cause orange and brown rust of sugarcane, respectively. Puccinia kuehnii has been confirmed in Asia, Australia and recently, the Caribbean basin, whereas P. melanocephala is distributed among the majority of sugarcane growing regions. Differentiating these two economically significant pathogens visually is problematic and limited to material exhibiting mature disease symptoms or spores. Partial ITS1, ITS2 and complete 5·8S sequences were generated from P. kuehnii and P. melanocephala isolates from around the world. PCR primers and dual labelled hydrolysis probes were designed for each pathogen for use in real‐time PCR and optimized using locked nucleic acids (LNA). The primers amplified DNA from their target pathogens and not from other species of Puccinia or fungal species isolated from sugarcane leaves. Optimized real‐time PCR conditions allowed the detection of 0·19 pg of P. kuehnii or P. melanocephala genomic DNA and differentiated the pathogens on sugarcane leaves prior to observing typical symptoms in the field. Primer‐introduced restriction analysis‐PCR (PIRA‐PCR) was used to detect a single nucleotide polymorphism (Pk ITS1 183A>G) in ITS1 of P. kuehnii. Allele 183A was observed in all samples, whereas 183G was detected in 52% of samples from Asia and Australia yet absent from all Caribbean basin samples. Long distance spore dispersal, dispersal through an intermediate location or improper movement of contaminated material could explain the introduction of P. kuehnii to the Western hemisphere. However, the current proliferation of the pathogen in the Americas is limited to isolates which contain only the 183A allele.     

Lateral roots of carrot have a low impact on alloinfections in cavity spot epidemic caused by <Emphasis Type="Italic">Pythium violae</Emphasis>

Carrot cavity spot, caused by a complex of Pythium species, is characterized by sunken elliptical lesions on the taproot. Recent epidemiological studies of P. violae have demonstrated the occurrence of both primary and secondary infections, with two types of secondary infection, autoinfection and alloinfection. Investigating the mechanisms underlying alloinfection and the role of carrot lateral roots, we asked whether direct physical root contact plays a role in alloinfection and whether root exudates enhance mycelial growth in soil alone. A rhizobox system was designed to differentiate the effects of each mechanism: a buffer zone created by nylon mesh was used to test the first mechanism, and young carrots with a root system similar to lateral roots were used to test the second. Alloinfections were generated in rhizoboxes via diseased taproots transplanted close to healthy, mature carrots. The nylon mesh had no significant effect on disease intensity (reflecting alloinfection), providing evidence that mycelial growth in soil contributed more to disease spread than did physical contact among roots. Nor did young carrots significantly affect alloinfection; thus root exudates had little effect on mycelial growth.     

Detection of Plasmodiophora Brassicae By PCR in Naturally Infested Soils

A nested polymerase chain reaction (PCR) method was developed for detection of DNA from Plasmodiophora brassicae in naturally infested field soil samples. The target sequences 389 bp and 507 bp were amplified from Swedish populations of P. brassicae. The protocols described enabled detection of DNA in various soil classes with an inoculum level of P. brassicae corresponding to a disease severity index (DSI) higher than 21 in a greenhouse bioassay. Three sequenced Swedish P. brassicae isolates had identical sequence in the 18S/ITS 1 region, but differed by a few nucleotides from an isolate sequenced in the UK. The results indicate that the primers used are general for P. brassicae, and consequently the nested PCR assay has a potential to be developed as a routine diagnostic test.     

A Phytophthora conserved transposon‐like DNA element as a potential target for soyabean root rot disease diagnosis

A transposon‐like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon‐like sequence. A 267‐bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real‐time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer‐based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.     

Molecular characterization of <Emphasis Type="Italic">Pythium</Emphasis> group F isolates by ribosomal-and intermicrosatellite-DNA regions analysis

Pythium group F is a ubiquitous, though minor, pathogen in several soilless and soil cultures; investigations were carried out to analyze different regions of the DNA and better understand the nature of this group. Fourty-two isolates were obtained from a variety of plants (cucumber, lettuce, tomato) grown in soil or soilless cultures collected in various countries (Canada, Denmark, France, Norway, Sweden and United Kingdom). All Pythium group F isolates displayed amplified ITS1-5,8S-ITS2 ribosomal DNA region (rDNA) of similar length, whereas polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) revealed that, among the seven enzymes used, polymorphism was only identified with Hin6I. After cloning of ITS1-5,8S-ITS2 rDNA region from Pythium group F isolates that displayed restriction polymorphism patterns with Hin6I, comparisons of sequence and restriction mapping data showed a slight variation consisting in a single base change. Inter Simple Sequence Repeat (ISSR)-PCR method was also used to obtain data related to the entire genome and not only to a single DNA region. It identified repeated motifs in the genome of Pythium group F isolates. Two primers (CAC)₅ and (CCA)₅ detected polymorphism, and isolates were classified among 11 molecular clusters. The genetic diversity of this group was not correlated with the geographical locations or the host plants from which the isolates originated. Polymorphism of Pythium group F isolates pointed out by ISSR is discussed     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

Specific and Sensitive Detection of Phytophthora nicotianae By Simple and Nested-PCR

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.     

Pathogenicity and fungicide sensitivity of Pythium and Phytopythium spp. associated with soybean in the Huang-Huai region of China

Pythium and Phytopythium spp. cause seed decay, damping off, and root rot in soybean, wheat, and many other crops. However, their diversity and importance as pathogens, particularly in different crop rotation systems, are largely unknown. A survey was conducted in the Huang-Huai region, one of the main areas of soybean–wheat rotation farming in China. In 2016–2018, we collected 300 soybean seedlings and 150 field soil samples from several representative locations, and identified 26 Pythium and 6 Phytopythium spp. from 212 isolates, based on internal transcribed spacer 2 (ITS2) and cytochrome oxidase subunit 1 sequences. The pathogenicity of these isolates was evaluated by growing soybean and wheat seeds in dishes and pots containing oomycete cultures. We found that 12 Pythium spp. (but no Phytopythium spp.) showed high pathogenicity on soybean and/or wheat, and nine of them (75%) were highly pathogenic on both crops. Among the nine species, Pythium spinosum, Pythium ultimum, Pythium species 1 (tentatively designated as ‘Candidatus Pythium huanghuaiense’), Pythium aphanidermatum, and Pythium myriotylum were highly abundant among all isolates (15%, 10%, 9%, 8%, and 5%, respectively). Nine species were selected for testing of sensitivity to the fungicides metalaxyl and mefenoxam. The EC₅₀ values were all less than 10 μg/ml, indicating little resistance. Minimum inhibitory concentration values indicated isolates were about twice as sensitive to mefenoxam as to metalaxyl. These results provide a systematic understanding of Pythium and Phytopythium species associated with soybean in the Huang-Huai region, which is important for disease management and breeding programmes.     

Detection and prediction of post harvest carrot diseases

Specific PCR primers were developed for identifying two post harvest pathogens, Mycocentrospora acerina and Fibularhizoctonia carotae, which cause liquorice rot and crater rot respectively, during prolonged low temperature storage of carrots. The methods allow routine detection of less than 0.3 pg of M. acerina DNA and less than 0.03 pg F. carotae DNA, even in the presence of large excess of plant or soil DNA. Standard PCR and quantitative PCR gave similar results and either method could be used in a practical situation. Experiments were carried out testing these methods on different types of carrot tissue- and soil- samples. Soil was sampled before sowing, and soil adhering to the roots or root tissue was sampled at different times during the growing season or at harvest. Soil adhering to the carrots at harvest had the best predictive ability for liquorice rot development during storage (R² predicted 74.9% using standard PCR), but samples taken during the growing season also gave reasonably good predictive ability values. PCR data from soil samples taken in the spring were not as good as a predictor for this disease. A dense sampling strategy using 20 m between sampling points generally gave better correlation between PCR data and disease data than using 40 m between the sampling points. Use of the developed methods in an IPM strategy for liquorice rot is discussed. For crater rot the correlation between PCR data and disease data was generally poor for all types of samples. These results are discussed in relation to the biology of F. carotae.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Development of real-time PCR assay using TaqMan probe for detection and quantification of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> in plant and soil

We developed real-time PCR assays using TaqMan probes to detect and quantify Rosellinia necatrix, the causal agent of white root rot in many plant species. Two sets of PCR primers and TaqMan probe indicated that their detection limits could be as low as 1 fg of template DNA. Using the real-time PCR assays with the TaqMan probes, we were able to quantify R. necatrix DNA in naturally diseased roots of Japanese pear and in artificially infested soil samples. Although the new assays were inadequate for use with naturally infested soil samples, nested PCR procedures improved the detectability of the new assays.     

Identity and variability of Pythium species associated with yield decline in aerobic rice cultivation in the Philippines

The cultivation of aerobic rice in the tropics enables farmers to save water without lowering productivity. Unfortunately, this system suffers from declining yields due to a disease complex involving nematodes, pathogenic Pythium spp. and nutrient deficiencies. Assessing the impact of each underlying factor can contribute to efficient disease control measures. This study therefore investigated pathogenic and genotypic variability among Pythium species from affected aerobic rice fields in the Philippines using pathogenicity assays and sequence information from the internal transcribed spacer (ITS) region and β‐tubulin gene. Three closely related Pythium spp., P. arrhenomanes, P. graminicola and P. inflatum, were recovered from affected aerobic rice fields. All P. arrhenomanes isolates reduced rice seedling growth, whereas only a few P. graminicola isolates and no P. inflatum isolates were pathogenic, indicating that P. arrhenomanes is probably the most important species affecting rice. Both P. arrhenomanes and P. graminicola isolates showed little genetic variation, despite the observed pathogenic variation within P. graminicola. Intraspecific variation was higher among P. inflatum isolates, but again no correlation was observed with phenotype. When screening P. arrhenomanes isolates from other hosts such as sugarcane, maize and several grasses, a link between pathogenic and genetic variability was detected. However, rice and maize isolates seemed to lack host specificity, and therefore crop rotation with maize might be a risky strategy to manage yield decline in Philippine aerobic rice fields.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum

The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.     

Development of a polyvalent RT‐PCR detection assay covering the genetic diversity of Cherry capillovirus A

The variability of Cherry capillovirus A (CVA) was analysed using a short, 275‐bp region of the viral RNA‐dependent RNA polymerase gene amplified by a polyvalent RT‐PCR assay. As for other members of the family Betaflexiviridae, CVA appears to show significant diversity, with an average pairwise nucleotide divergence of 9·4% between isolates in the analysed region. Phylogenetic analyses provide evidence for the existence of at least five clusters of CVA isolates, one of which is associated with noncherry hosts of the virus, providing evidence that transmission of CVA isolates between cherry and noncherry hosts is probably rare. Comparison of existing detection techniques using a panel of CVA isolates representative of the various phylogenetic groups indicated that dot‐blot hybridization assays show high polyvalence but may lack the sensitivity to detect CVA in some samples. On the other hand, available detection primers failed to amplify a wide range of CVA isolates. Partial genome sequencing of two divergent isolates allowed the identification of conserved genomic regions and the design of new primer pairs with improved polyvalence. These new primer pairs were used to develop PCR assays allowing the reliable detection of CVA isolates belonging to all phylogenetic clusters.     

A simple detached leaf assay provides rapid and inexpensive determination of pathogenicity of Pythium isolates to ‘all year round’ (AYR) chrysanthemum roots

A detached leaf assay was developed to determine the pathogenicity of Pythium isolates to cut‐flower chrysanthemum roots. Leaves from young plants were excised and inoculated by insertion of a plug of mycelium into a slit cut in the excised petiole. After incubation leaves were assessed for presence and extent of necrosis. Necrosis indicated pathogenicity and was consistently confirmed by comparisons with whole plant inoculations. The rate of necrosis spread also gave some indication of virulence. Isolates of Pythium sylvaticum, P. ultimum and HS group were the most virulent, with a mean rate of spread of 14·6 mm per day, significantly (P < 0·05) faster than the mean rate of spread, 1·6 mm per day, of less virulent isolates. Less virulent isolates included P. irregulare, P. oligandrum and P. aphanidermatum. The latter was unexpected, as P. aphanidermatum is an important species in pythium root rot epidemics in chrysanthemums elsewhere. The value of the detached leaf assay for screening large numbers of isolates was demonstrated in a survey of isolates from clinic samples from chrysanthemum nurseries and in a series of dilution‐plating experiments looking at numbers of Pythium propagules in commercial chrysanthemum beds showing root rot. In the survey, the predominant pathogenic species was identified as P. sylvaticum and the most likely source of infection was contaminated soil as opposed to blocking media or irrigation water, whilst in soil colonization studies the use of detached leaf assays demonstrated a relationship between pathogenic inoculum concentration in soil and the expression of root rot symptoms.     

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.     

建兰胶孢炭疽菌ITS序列分析及其PCR快速检测

由胶孢炭疽菌Colletotrichum gloeosporioides引起的炭疽病是建兰的重要病害.为建立快速检测该病原菌的方法,以ITSl/ITS4为引物,对15个建兰胶孢炭疽菌的ITS进行PCR扩增及测序,将测定的序列与炭疽菌属其它种的ITS序列进行比对分析,设计特异性引物CFl/CR1,并通过常规和巢式PCR对建兰胶孢炭疽菌进行检测.结果显示,15个菌株中有13个菌株ITS序列与该菌的模式种序列相似性高达99％以上,而另外2个菌株相似性则为86％;供试菌株在系统发育树上聚为2个不同的分支;引物CFl/CR1通过常规PCR可从1 ng的建兰胶孢炭疽菌基因组DNA中扩增到目的条带,而利用引物ITSl/ITS4和CF1/CR1通过巢式PCR可从1 pg的基因组DNA中扩增到目的条带,即巢式PCR反应检测灵敏度较常规PCR至少高1 000倍.表明建立的巢式PCR法可从自然感病的建兰叶片组织中检测到胶孢炭疽菌.     

Prevalence of Berkeleyomyces basicola infections in black rot-affected carrot determined using the MCM7 gene region

Thielaviopsis and related taxa are responsible for losses in several agricultural crops. In carrot these pathogens can cause black rot, mainly after harvesting. Molecular studies using species-specific DNA sequences are the principal tools for accurate identification of these pathogens. The objective of this work was to investigate which taxa are associated with black rot in carrot in Brazil using gene markers and Bayesian phylogenetic analysis. Eighteen isolates were obtained from carrots with symptoms of black rot. Through morphological characteristics all isolates were classified as Thielaviopsis-like. Bayesian inference using only MCM7 sequences and a concatenated data set (LSU, MCM7 and 60S rRNA) allowed identification of all isolates as belonging to Berkeleyomyces basicola. All isolates induced black rot in carrot. Although our studies include a small number of isolates, the results indicate the likely predominance of B. basicola in causing black rot in carrot in Brazil, and that MCM7 sequences are enough to distinguish the Berkeleyomyces species by Bayesian inference.