Differential Cultivars Research Articles

A systematic approach for identifying unique genomic sequences for Fusarium oxysporum f. sp. lactucae race 1 and development of molecular diagnostic tools

Fusarium oxysporum f. sp. lactucae (FOLac) is a soil- and seedborne fungal pathogen that causes Fusarium wilt of lettuce, an important disease threatening global lettuce production. Based on pathogenicity on differential lettuce cultivars, four races (1-4) have been identified, with race 1 the only race detected in the United States, and the closely related, emerging race 4 known only in Europe. The development of race-specific diagnostic tools is hindered by insufficient genomic data to distinguish between the two races and FOLac from other F. oxysporum formae speciales and nonpathogenic isolates. Here, we describe a systematic approach for developing diagnostic markers for FOLac race 1 that utilized a comprehensive sequence database of F. oxysporum to identify 15 unique genomic sequences. Marker specificity was validated through an exhaustive screening process against genomic data from 797 F. oxysporum isolates representing 64 formae speciales and various plants and non-plant substrates. One of the unique sequences was used to develop a TaqMan quantitative polymerase chain reaction assay and a recombinase polymerase amplification assay, both exhibiting 100% sensitivity and specificity when tested against purified DNA from 171 F. oxysporum isolates and 69 lettuce samples. The relationship between qPCR Ct values and colony forming units (CFU)/g values was also determined. This study not only introduces a new marker for FOLac race 1 diagnostics and soil quantitation, but also underscores the value of an extensive genomic database and screening software pipeline for developing molecular diagnostics for F. oxysporum formae speciales and other fungal taxa.

Detection of novel pathogenic variants of Fusarium oxysporum f. sp. lactucae in California

AbstractFusarium wilt, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. lactucae (FOL), is an economically important disease of lettuce (Lactuca sativa) throughout the world. Four pathogenic races of FOL are reported, though only race 1 is known to exist in the United States. Recently, Californian lettuce growers have expressed increasing concern about Fusarium wilt, as some race 1‐resistant cultivars have exhibited susceptibility, and some susceptible cultivars have displayed reduced disease severity. To determine whether such changes in disease patterns are responses to potentially novel variants, we collected infected plants from commercial fields in the Salinas Valley and Santa Maria regions, isolated the fungus, and conducted a series of pathogenicity tests using a set of FOL race differential cultivars over 2 years (2022 and 2023) in controlled conditions. Pathogenicity tests revealed two new FOL variants that elicited novel disease reaction patterns on the set of differential cultivars that have not been previously described. Isolates Fol621 and Fol621s were less aggressive on race 1‐susceptible Banchu Red Fire, designated Variant‐1. Isolate VSP‐0916 incited severe Fusarium wilt on race 1‐resistant Costa Rica No. 4, designated Variant‐2. Moreover, VSP‐0916 exhibited high aggressiveness and the ability to induce disease in other race 1‐resistant cultivars. This study is the first documentation, to our knowledge, of the occurrence of FOL‐inciting Fusarium wilt on race 1‐resistant lettuce in the United States. Our work provides valuable information critical for the development of Fusarium wilt management strategies, including broad‐spectrum resistance breeding efforts against multiple FOL races.

Molecular and Morphological Identification of Sarocladium Species Causing Sheath Rot of Rice in Thailand and Their Division into Physiological Races.

Sheath rot and dirty panicle are some of the major diseases of rice in Thailand. The diseases are traditionally considered to be caused by the pathogen Sarocladium oryzae and damage and lower both the quantity and quality of rice grain. In this study, 32 fungal isolates collected from the central and northeastern regions of Thailand were analysed phylogenetically using three molecular markers (ITS, D1/D2 of 28S rDNA and ACT) and physiological races were determined on 10 differential rice cultivars. We found that S. oryzae is not the only causal agent of sheath rot in Thailand, but S. attenuatum was also found. Despite having similar morphological features, the phylogenetic analysis recognised 11 of 32 isolates as S. attenuatum and the remaining isolates as S. oryzae. This is the first report of S. attenuatum causing sheath rot of rice in Thailand in addition to S. oryzae. Evaluation of physiological races revealed high pathogenic diversity of the two species. Thus, 16 and 11 physiological races were recorded from 21 isolates of S. oryzae and 11 isolates of S. attenuatum, respectively. These results indicate that both S. oryzae and S. attenuatum are the causal agents of rice sheath rot and dirty panicle in Thailand and that they are pathologically diverse.

Constructed Rice Tracers Identify the Major Virulent Transcription Activator-Like Effectors of the Bacterial Leaf Blight Pathogen

Xanthomonas oryzae pv. oryzae (Xoo) injects major transcription activator-like effectors (TALEs) into plant cells to activate susceptibility (S) genes for promoting bacterial leaf blight in rice. Numerous resistance (R) genes have been used to construct differential cultivars of rice to identify races of Xoo, but the S genes were rarely considered. Different edited lines of rice cv. Kitaake were constructed using CRISPR/Cas9 gene-editing, including single, double and triple edits in the effector-binding elements (EBEs) located in the promoters of rice S genes OsSWEET11a, OsSWEET13 and OsSWEET14. The near-isogenic lines (NILs) were used as tracers to detect major TALEs (PthXo1, PthXo2, PthXo3 and their variants) in 50 Xoo strains. The pathotypes produced on the tracers determined six major TALE types in the 50 Xoo strains. The presence of the major TALEs in Xoo strains was consistent with the expression of S genes in the tracers, and it was also by known genome sequences. The EBE editing had little effect on agronomic traits, which was conducive to balancing yield and resistance. The rice-tracers generated here provide a valuable tool to track major TALEs of Xoo in Asia which then shows what rice cultivars are needed to combat Xoo in the field.

Virulence diversity of the yellow rust pathogen population in Dagestan

Background. Yellow rust incidence on Triticum L. has been observed to surge up in Dagestan in recent years, facilitating selection of highly resistant genotypes. Information on the effectiveness of Yr genes and the pathogen’s virulence and racial composition is needed to interpret field assessment data correctly. The objective of this study was to describe the virulence of the Dagestan population of Puccinia striiformis West. at the seedling stage, evaluate the resistance of differentiators to yellow rust in the field, and identify invasive PstS1 and PstS2 races.Materials and methods. Leaves with urediniopustules were collected from bread wheat accessions at Dagestan Experiment Station of VIR (DЕS VIR) in 2020–2022. Virulence was analyzed in the laboratory using 14 AvYr lines (Avocet NIL) and 15 differential wheat cultivars. Age-specific resistance of virulence testers under high natural infection pressure was studied in the field of DES VIR. A set of SCAR markers (SCP19M24a1, SCP19M24a2, SCP19M26a1, and SCP19M26a2) were used to identify invasive races.Results and discussion. Avocet lines with Yr5, Yr10, Yr15, Yr24, and Yr26, and cv. ‘Moro’ (Yr10, YrMor) manifested resistance to all isolates. Virulence to Yr17 was detected only in the isolates from cvs. ‘Graf’ and ‘Svarog’ carrying this gene, and ‘Siete Ceros’. The Dagestan collection demonstrated high genetic diversity. Common phenotypes were identified on cv. ‘Graf’ in 2021 and 2020. There was no temporal differentiation between pathogen collections in the years of testing. High field resistance to yellow rust was recorded in lines with Yr5, Yr8, Yr10, Yr15 and Yr24, and in cvs. ‘Moro’, ‘Compair’, ‘Carstens’ and ‘Spaldings Prolific’. Cvs. ‘Reichersberg 42’ and ‘Heines Peko Vilmorin 23’ were resistant in 2020 and 2022, and moderately affected (up to 10%) in 2021. Molecular analysis identified all three isolates belonging to the invasive PstS2 group on cv. ‘Siete Cerros’ and L 650 in 2021, and cv. ‘Vassa’ in 2022.

Automating high-throughput screening for anthracnose resistance in common bean using allele specific PCR

BackgroundCommon beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results.ResultsThe 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-42 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time.ConclusionResults suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools.

Molecular Marker Development for the Rapid Differentiation of Black Rot Causing Xanthomonas campestris pv. campestris Race 7.

Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of Brassica crops that causes black rot disease throughout the world. At present, 11 physiological races of Xcc (races 1-11) have been reported. The conventional method of using differential cultivars for Xcc race detection is not accurate and it is laborious and time-consuming. Therefore, the development of specific molecular markers has been used as a substitute tool because it offers an accurate and reliable result, particularly a quick diagnosis of Xcc races. Previously, our laboratory has successfully developed race-specific molecular markers for Xcc races 1-6. In this study, specific molecular markers to identify Xcc race 7 have been developed. In the course of study, whole genome sequences of several Xcc races, X. campestris pv. incanae, X. campestris pv. raphani, and X. campestris pv. vesicatoria were aligned to identify variable regions like sequence-characterized amplified regions and insertions and deletions specific to race 7. Primer pairs were designed targeting these regions and validated against 22 samples. The polymerase chain reaction analysis revealed that three primer pairs specifically amplified the DNA fragment corresponding to race 7. The obtained finding clearly demonstrates the efficiency of the newly developed markers in accurately detecting Xcc race 7 among the other races. These results indicated that the newly developed marker can successfully and rapidly detect Xcc race 7 from other races. This study represents the first report on the successful development of specific molecular markers for Xcc race 7.

POSTULATION OF YR RESISTANCE GENES TO STRIPE RUST IN 12 COMMERCIAL WHEAT CULTIVARS OF RUSSIAN BREEDING

Stripe rust (Puccinia striiformis f. sp. tritici) is a common wheat disease of economic importance in all world wheat production regions. In most of the regions stripe rust causes yield losses of 10-70 %, depending on the time infection occur, host susceptibility, disease development, and the duration of the epidemics. In South Russia even in unfavourable conditions for the pathogen, stripe rust occurred in the region every year, and in some areas there are foci of the disease with of up to 50% severity.
 Phytopathological testing is one of the least expensive and most effective methods for the rapid identification of juvenile resistance genes in varieties against pathogens. It allows to obtain detailed information about the genetics of the host and pathogen in a short time. In the research Yr resistant genes and its combinations were postulated in winter wheat varieties of Russian breeding to North-Caucasus population of stripe rust by this method.
 Purpose of this study was identification of known stripe rust resistance genes in 12 commercial winter wheat cultivars by phytopathological testing.
 Material and methods: The postulation of known resistance genes was carried out in a greenhouse complex (seedling stage) and at the research station (adult plant stage) of the Laboratory of Plant Immunity to Diseases of the Federal Scientific Center for Biological Plant Protection (Krasnodar).
 For postulation, we chose the method by Dubin et al. (1989), based on the comparison of response types of differential cultivars and studied varieties to Pst isolates.
 Results. In 10 out of 12 winter wheat varieties of Russian breeding, 13 stripe rust resistance genes and their combinations were postulated.: Yr3, Yr3a, Yr6, Yr32, Yr2+6, Yr2+9, Yr39+Alp, Yr4+12, Yr4b, Yr3a+4a+H46, YrA, YrSp, YrSU. The following resistance genes have not been identified: Yr2, Yr5, Yr10, Yr10+Mor, Yr24, Yr27, Yr25+32, since no isolates virulent to carriers of these genes were isolated from the North Caucasian population of P. striiformis; and they are highly effective against this population. Highly effective (Yr3, Yr2+9, Yr3a, Yr4+12, Yr3a+4a+H46) resistance genes were identified in the Kurs, Morozko, and Step’ varieties. We note a combination of effective Yr2+6 genes in the Gurt variety.
 In field conditions, high resistance (degree of damage up to 10%) to the North Caucasian population of the stripe rust pathogen was shown by lines and differential cultivars containing the following resistance genes: Yr2, Yr3, Yr8, Yr10, Yr24, Yr25, Yr27, Yr10+Mor, Yr3a, Yr25+32, Yr3a+4a+H46, Yr3с+Min, Yr4+12, Yr7+25, Yr8+19, 8+6+25, YrSp. Due to the low degree of damage, these genes and their combinations are classified as highly effective and can be involved in the breeding process to create new resistant varieties. Varieties containing these genes can also be recommended for breeders and used in production for effective disease control.

Race Structure and Molecular Diversity of Colletotrichum lindemuthianum of Common Bean in Zambia.

Anthracnose, caused by the fungus Colletotrichum lindemuthianum, is a major disease of common bean (Phaseolus vulgaris L.) worldwide. C. lindemuthianum is genetically highly variable, and understanding the pathogen's diversity and distribution is a key step in developing common bean varieties with durable anthracnose resistance. The objectives of this study were to (i) characterize the race structure of C. lindemuthianum in Zambia and (ii) assess the molecular diversity of C. lindemuthianum in Zambia. A field survey was conducted in 20 bean-growing districts in Zambia to collect anthracnose symptomatic bean plants. A total of 103 C. lindemuthianum isolates were collected and characterized based on their reactions on 12 common bean race differential cultivars. RAM and ERIC-BOX DNA markers were used to assess molecular diversity of 60 isolates. A total of 58 races were characterized from the 103 isolates. Race 5 was the least virulent, and race 1631 was the most virulent based on their reaction on the 12 race differential cultivars. Race 19 had the highest recovery frequency (11%) and was the most extensively dispersed among the 22 bean-growing districts from where the isolates were collected. Only six races had previously been reported in Zambia, and 52 races were identified as new races reported for the first time in Zambia. Two races were virulent only on Andean cultivars, 11 races were virulent only on Middle American cultivars, and 45 races were virulent on both Andean and Middle American cultivars. No individual isolate showed pathogenicity on all the differential cultivars, and no isolate overcame the Co-4, Co-5, and Co-7 resistance gene pyramid that naturally exists in G2333. Phylogenetic analysis categorized the 60 isolates in six major clusters and six subclusters. The 60 isolates showed high genetic heterogeneity among and within a race of the same virulence. The study has revealed the existence of both Andean and Middle American races and extensive molecular diversity of C. lindemuthianum in Zambia. The knowledge on the race structure of C. lindemuthianum that this study has provided will be valuable for making breeding decisions on the host plant resistance genes required for developing common bean varieties with durable resistance to anthracnose in Zambia.

Characterization of Uromyces appendiculatus First Races in Sri Lanka and Identification of Genes for the Development of Rust-Resistant Snap Beans.

Bean rust is a devastating disease of snap beans in Sri Lanka. Our study endeavored to develop snap beans with resistance to Uromyces appendiculatus, the bean rust pathogen reported to have numerous virulent races. Accordingly, we needed a detailed understanding of the virulence of U. appendiculatus and to identify genes conferring effective rust resistance. To investigate the virulence, we collected leaves of snap bean with rust symptoms from four snap bean producing districts of Sri Lanka. From these samples we established 57 single pustule isolates. Each isolate was inoculated on a set of 12 differential cultivars, six from the Andean and six from the Middle American gene pools of common bean. The virulence of the isolates segregated into four distinct groups that comprised the first four reported races of U. appendiculatus in Sri Lanka, 23-5, 31-1, 31-11, and 63-21. All races were significantly more virulent on the Andean than on the Middle American differential cultivars. PI 1819966, a Middle American differential cultivar containing the Ur-11 gene, conferred resistance to all four races. Three other Middle American cultivars were resistant to three races. Conversely, five Andean differential cultivars were susceptible to all four races. PI 160418 was the only Andean differential cultivar with resistance to three races. The results suggested that rust resistance genes from the Middle American differential cultivars could be combined in different ways with the resistance of Andean PI 260418 to develop snap bean varieties with broad resistance to all races of U. appendiculatus in Sri Lanka.

Primer reporte de Fusarium oxysporum f. sp. niveum raza 1 como agente causal de la marchitez vascular de la sandía en México

Vascular wilt (Fusarium oxysporum f. sp. niveum [Fon]) is the main fungal disease of watermelon crops worldwide and can be caused by any of the four known races of this fungus (race 0, 1, 2 and 3). In Mexico, there are no reports of the presence of any of these races. The presence of plants with wilt and vascular necrosis has been observed in commercial watermelon plantations in the municipality of Hermosillo, Sonora, Mexico, mainly during the spring-summer growing seasons. The aim of this study was to identify the Fon race causing vascular wilt in watermelon plants in Hermosillo, Sonora. Three fungal isolates were collected from symptomatic plants. Based on their morphological characteristics, DNA amplification of the isolates (with specific primers Fon-1/Fon-2, FONSIX6F/FONSIX6R and FNR3-F/FNR3-R, which allow differentiating Fon race 1, 2 and 3) and inoculation into three differential watermelon cultivars, the three isolates (UESFON01, UESFON02 and UESFON03) were identified as F. o. f. sp. niveum race 1. To our knowledge, this is the first report where F. o. f. sp. niveum race 1 is identified as the causal agent of wilt, vascular necrosis and plant death in watermelon crops in Mexico.

First Report of Fusarium oxysporum f. sp. lactucae Race 4 Causing Lettuce Wilt in Spain.

Spain is the fourth largest lettuce-producing country in the world and the leading European producer. Much of the production, mainly grow in open field, is dedicated to export with a value of 887 million U.S. dollars per year. In summer 2021 wilting symptoms were observed in a commercial field crop on butterhead lettuce 'Amible' in Albacete (Castilla-La Mancha, Spain). Approximately 15% of plants were affected, but losses were even more severe on subsequent crops. Vascular tissue of affected plants showed a brown to red discoloration. Sections of infected vascular tissue (3 to 5 mm long) were surface sterilized in 70% ethanol for 30 s, washed three times with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate (100 mg/liter). From 5-day-old cultures typical pale cream to purplish mycelia with microconidia, macroconidia, and chlamydospores of Fusarium oxysporum were observed. Microconidia were abundant on carnation leaf agar and measured 6.1 to 9.2 μm (mean 7.1 ± 0.7 μm; n=50)). Macroconidia were sparse, three-septate, straight to slightly curved, 23.3 to 34.8 × 4 to 5.2 µm (mean 31.5 ± 2.8 × 4.2 ± 0.3 µm; n=50). Chlamydospores were terminal and intercalary, rough walled, and measured 7.2 to 10.1 μm (mean 9.5 ± 0.6 μm; n=50) µm. DNA was extracted from three single-spore isolates using the protocol of Querol et al. (1992) and the translation elongation factor 1-α gene (TEF) was sequenced with exTEF-F/FUexTEF-R primers as described by Taylor et al. (2016). All TEF sequences (GenBank accession no. OP903519) were identical. In BLAST analyses, the isolates showed 100% identity to the corresponding region of Fusarium oxysporum f. sp. lactucae (FOL) race 4 (MK059958). All Spanish isolates were identified as FOL race 4 using a race-specific polymerase chain reaction (PCR) with the primers FPUF/FPUR (Gilardi et al. 2017), and a previously identified FOL race 4 isolate Fus 1.01 as a positive control. Pathogenicity tests were conducted to confirm the positive result of the race 4-specific PCR and to complete Koch's postulates. Three differential lettuce cultivars ('Costa Rica No. 4', 'Banchu Red Fire', and 'Romana Romabella 30 CN') provided by Rijk Zwaan (The Netherlands) were inoculated with three Spanish isolates (Al1A1, Al1D, Al2B) and the Fus 1.01 isolate used as FOL race 4 positive control (Claerbout et al., 2018). Roots of 3-week-old plants (five replicates per treatment) were dipped in a spore suspension (1 × 106 conidia/ml) for 10 min before transplanting into 250-ml pots with sterile substrate. Non-inoculated control plants were dipped in sterile water for 10 min. The experiment was carried out twice. Inoculated lettuce seedlings were planted and maintained in a growth chamber (25°C day, 18°C night). Plants were slightly watered every other day. After 21 days, wilting was observed in the cultivars 'Costa Rica No. 4' and 'Romana Romabella 30 CN'. Moreover, taproots were cut longitudinally, and vascular browning was observed in the taproot. No discoloration could be observed in the taproot of 'Banchu Red Fire' plants, coinciding with the result of isolate Fus 1.01 used as FOL race 4 positive control. Non-inoculated control plants remained healthy and vascular browning was not observed. In both experiments, F. oxysporum f.sp. lactucae was consistently reisolated using PDA medium. These results confirmed that the isolates Al1A1, Al1D and Al2B were FOL race 4. This race has recently been identified in The Netherlands (Gilardi et al. 2017), Belgium (Claerbout et al. 2018), United Kingdom, Ireland (Taylor et al. 2019) and Italy (Gilardi et al. 2019) and could become a serious threat to Spain lettuce production.

First report of Fusarium oxysporum f. sp. lactucae Race 1 causing lettuce wilt in Northern Ireland.

In the UK, lettuce is produced both in the field and in greenhouses or polytunnels. In summer 2022, wilt symptoms were observed for the first time on lettuce (cv. Amica) grown in soil in part of a single 0.55 ha greenhouse in County Armagh, Northern Ireland (NI). Initial presentation of symptoms was stunting of plants, followed by wilting and yellowing of lower leaves in approx. 12% of the plants. Orange-brown discoloration of vascular tissue in the tap root of affected plants was also observed. To isolate the causal pathogen, sections (0.5 cm2) of symptomatic vascular tissue from 5 plants were surface sterilized in 70% ethanol for 45 s, washed twice in sterile water and placed on potato dextrose agar (PDA) amended with 20 µg mL-1 chlortetracycline. Plates were incubated at 20°C for 5 days and fungal colonies sub cultured onto PDA. Isolates from all five samples displayed morphology typical of Fusarium oxysporum and were cream to purple in colour with abundant microconidia and occasional macroconidia. DNA was extracted from 5 isolates and part of the translation elongation factor 1- α (EF1-α) gene amplified by PCR and sequenced as described by Taylor et al. (2016). All EF1-α sequences were identical (OQ241898) and matched with F. oxysporum f. sp. lactucae race 1 (MW316853.1, isolate 231274) and race 4 (MK059958.1, isolate IRE1) with 100% sequence identity using BLAST. Isolates were then identified as FOL race 1 (FOL1) using a race-specific PCR assay (Pasquali et al. 2007). Next, pathogenicity and race identity of one isolate (AJ773) was confirmed using a set of differential lettuce cultivars (Gilardi et al. 2017); Costa Rica No. 4 (CR; FOL1 resistant), Banchu Red Fire (BRF; FOL4 resistant) and Gisela (GI; FOL1 / FOL4 susceptible). Plants were inoculated with AJ773 (this study), ATCCMya-3040 (FOL1, Italy; Gilardi et al. 2017) and LANCS1 (FOL4, UK; Taylor et al., 2019). Roots of 16-day-old lettuce plants (8 replicates per cultivar/isolate) were trimmed and soaked in a spore suspension (1 x 106 conidia mL-1) for 10 min before transplanting into compost in 9 cm pots. Control plants for each cultivar were dipped in sterile water. Pots were placed in a glasshouse (25C day, 18C night). Inoculation with AJ773 and FOL1 ATCCMya-3040 resulted in typical symptoms of Fusarium wilt for BRF and GI 12-15 days after inoculation, while for FOL4 LANCS1, wilting was observed in CR and GI. Thirty-two days after inoculation, plants were cut longitudinally and vascular browning was observed in all plants where wilt was present. All uninoculated control plants, CR inoculated with FOL1 ATCCMya-3040 or AJ773, and BRF inoculated with FOL4 LANCS1 remained healthy. These results confirm the identity of isolate AJ773 from NI as FOL1. Koch's postulates were fulfilled by consistent re-isolation of F. oxysporum from BRF and GI plants and identifying as FOL1 using the race-specific PCR. No FOL was re-isolated from control plants of any cultivar. Fusarium wilt in England and the Republic of Ireland first reported by Taylor et al. (2019) was identified as FOL4 and has been confined to indoor lettuce production with further outbreaks caused by the same race. However, FOL1 was recently identified in Norway in a soil-grown glasshouse crop (Herrero et al. 2021). The presence of both FOL1 and FOL4 in neighbouring countries within the UK is a serious risk to lettuce production and is of particular importance to growers who rely on knowledge of cultivar resistance to specific FOL races to make decisions on which variety to plant.

Virulence and genetic variability among Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara isolates from common bean (Phaseolus vulgaris L.)

Colletotrichum lindemuthianum is a fungus that causes bean anthracnose. This study analyzed the virulence and genetic variability among and within C. lindemuthianum isolates occurring in bean-growing areas of southern Ethiopia. Symptomatic common bean leaves and pods were collected from southern Ethiopia. Forty single-spore isolates obtained from the bean leaves and pods were used for this study. The isolates were grouped into 31 physiological races based on their responses to a differential set of common beans. Five of the physiological races (2047, 4031, 2052, 2064, 2174 and 2176) were extremely virulent, causing susceptible reactions on 10 to 11 different cultivars. Rep-polymerase chain reaction revealed high polymorphism, with an average of 87.5% polymorphic loci. The mean Na, Ne, He and I were 1.83, 1.465, 0.278 and 0.427, respectively, within C. lindemuthianum races. Analysis of molecular variance (AMOVA) partitioned a larger part of the total genetic variation within populations (80%). Our collection includes highly virulent races that infected the differential cultivars G2333, Perry marrow and Pi207262 as a possible source of resistance. The differential cultivar G2333 showed strong resistance and can be a potential source of resistance, while the differential cultivars Perry marrow and Pi207262 would be alternative donor parents as sources of resistance in breeding programs for the development of varieties with broader resistance. This study could be a scientific basis for disease prevention and for development of common bean cultivars resistant to bean anthracnose. Identification of a wider range of isolates must be carried out, collecting common bean in different seasons.

Identification of Fusarium oxysporum f. sp. lactucae Race 1 as the Causal Agent of Lettuce Fusarium Wilt in Greece, Commercial Cultivars' Susceptibility, and Temporal Expression of Defense-Related Genes.

Fusarium wilt of lettuce is found throughout the world, causing significant yield losses. Lettuce is the most-cultivated leafy vegetable in Greece, affected by a large number of foliar and soil-borne pathogens. In this study, 84 isolates of Fusarium oxysporum, obtained from soil-grown lettuce plants exhibiting wilt symptoms, were characterized as belonging to race 1 of F. oxysporum f. sp. lactucae based on sequence analysis of the translation elongation factor 1-alpha (TEF1-α) gene and the rDNA intergenic spacer (rDNA-IGS) region. The isolates were also assigned to one single race through PCR assays with specific primers targeting race 1 and race 4 of the pathogen. In addition, four representative isolates were confirmed to be associated with race 1 based on the pathogenicity tests with a set of differential lettuce cultivars. Artificial inoculations on the most commonly cultivated lettuce cultivars in Greece revealed that the tested cultivars varied regarding their susceptibility to F. oxysporum f. sp. lactucae race 1. Cultivars (cvs.) "Cencibel" and "Lugano" were found to be highly susceptible, while cvs. "Sandalina" and "Starfighter" were the most resistant ones. Expression analysis of 10 defense-related genes (PRB1, HPL1, LTC1, SOD, ERF1, PAL1, LOX, MPK, BG, and GST) was carried out on artificially inoculated lettuce plants of the four above cultivars at different time points after inoculation. In resistant cultivars, a higher induction rate was observed for all the tested genes in comparison with the susceptible ones. Moreover, in resistant cultivars, all genes except LTC1, MPK, and GST showed their highest induction levels in their earliest stages of infection. The results of this study are expected to contribute to the implementation of an integrated management program to control Fusarium wilt of lettuce, based mainly on the use of resistant cultivars.

Skim resequencing finely maps the downy mildew resistance loci RPF2 and RPF3 in spinach cultivars whale and Lazio.

Commercial production of spinach (Spinacia oleracea L.) is centered in California and Arizona in the US, where downy mildew caused by Peronospora effusa is the most destructive disease. Nineteen typical races of P. effusa have been reported to infect spinach, with 16 identified after 1990. The regular appearance of new pathogen races breaks the resistance gene introgressed in spinach. We attempted to map and delineate the RPF2 locus at a finer resolution, identify linked single nucleotide polymorphism (SNP) markers, and report candidate downy mildew resistance (R) genes. Progeny populations segregating for RPF2 locus derived from resistant differential cultivar Lazio were infected using race 5 of P. effusa and were used to study for genetic transmission and mapping analysis in this study. Association analysis performed with low coverage whole genome resequencing-generated SNP markers mapped the RPF2 locus between 0.47 to 1.46Mb of chromosome 3 with peak SNP (Chr3_1, 221, 009) showing a LOD value of 61.6 in the GLM model in TASSEL, which was within 1.08 Kb from Spo12821, a gene that encodes CC-NBS-LRR plant disease resistance protein. In addition, a combined analysis of progeny panels of Lazio and Whale segregating for RPF2 and RPF3 loci delineated the resistance section in chromosome 3 between 1.18-1.23 and 1.75-1.76Mb. This study provides valuable information on the RPF2 resistance region in the spinach cultivar Lazio compared to RPF3 loci in the cultivar Whale. The RPF2 and RPF3 specific SNP markers, plus the resistant genes reported here, could add value to breeding efforts to develop downy mildew resistant cultivars in the future.

Characterization of Podosphaera macularis on the Most Prevalent Hop Genotype in the Pacific Northwest

As of 2020, the hop cultivar ‘HBC 394’, trademarked name Citra brand, was the most widely planted hop cultivar in Washington, Oregon, and Idaho, with the acreage harvested in all three states nearly doubling from 2018 to 2020. This research characterized the pathogenic diversity of isolates of Podosphaera macularis on Citra brand and determined whether isolates with local or general adaption have emerged coincident with Citra brand becoming the most widely planted cultivar in the region. Among 27 isolates obtained from Citra brand, all could infect differential cultivars possessing the R-genes Rb, R3, and R5; nine isolates were also virulent on the differentials for R4 and R6. In a broader collection of 92 other isolates from Citra brand, 91 (98.9%) lacked R6 virulence. Inoculations of seven P. macularis isolates with complex virulence onto detached leaves of Citra brand all produced disease, indicating that a known major R-gene does not predict the powdery mildew phenotype in Citra brand. Citra brand-derived isolates and isolates representing the three predominant pathogenic races of P. macularis in the Pacific Northwest had no major difference in aggressiveness when inoculated on Citra brand leaves. Citra brand-derived isolates also caused disease levels on a range of hop cultivars similar to levels caused by isolates derived from other hop cultivars. These findings adduce that P. macularis isolates found on Citra brand may have varied phenotypes associated with known Pacific Northwest pathogen races. However, currently, isolates derived from Citra brand display neither local adaptation for increased virulence on Citra brand nor general adaptation for enhanced virulence on other cultivars. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Investigating phenotypic traits as potential drivers of the emergence of EU_37_A2, an invasive new lineage of Phytophthora infestans in Western Europe

AbstractSince the mid‐2010s, Phytophthora infestans clones that have been dominant in Western Europe from the beginning of the 21st century, for example, EU_13_A2, EU_6_A1 and EU_1_A1, are being replaced by several other emerging clones, including EU_37_A2. The objective of this study was to determine whether the main drivers for the success of EU_37_A2 in Western Europe are associated with decreased fungicide sensitivity, increased virulence and/or aggressiveness. Axenic P. infestans cultures were sampled in the 2016 and 2017 growing seasons from potato crops in France and the United Kingdom. Amongst these, four genotypes were identified: EU_37_A2, EU_13_A2, EU_1_A1 and EU_6_A1. Although a wide range of fluazinam sensitivity was found amongst individual isolates, clonal lines EU_13_A2 and EU_37_A2 showed decreased sensitivity to fluazinam. EU_37_A2 overcame the R5 differential cultivar more often than isolates of EU_1_A1 or EU_6_A1. However, this does not explain the competitive advantage of EU_37_A2 over the virulent EU_13_A2. The fittest genotype, as measured by aggressiveness under controlled conditions, was EU_6_A1, followed by EU_37_A2, EU_13_A2 and then EU_1_A1. EU_37_A2 isolates also showed a shorter latent period than either EU_6_A1 or EU_13_A2, which could favour its long‐term persistence. Overall, the data suggest that the emergence of EU_37_A2 in Western Europe was driven by its resistance to a then‐major fungicide and shorter generation time. This conclusion is further supported by the fact that EU_37_A2 emergence was slowed by the progressive reduction in the use of fluazinam as a single active ingredient in the years following its initial detection.

IDENTIFICATION AND CONTROL OF PATHOGENIC RACES OF Ustilago nuda LOOSE SMUT CAUSAL PATHOGEN OF BARLEY

Identification of Ustilago nuda (Jens.) Rostr., the causal agent of barley (Hordeum vulgare L.) loose smut disease using the Random Amplified Polymorphic (DNA) markers is faster and easier than using of the traditional methods (differential cultivars),where identify physio-logical races may needs several years. Also, at the two locations, Giza and Sakha Agricultural Research Stations, several effectors were tested to find out their effectiveness in ridding the grains of the fungus that resides inside them, i.e. (soaking in some natural oils, exposure to Gamma ray and dressing with some new fungicides). Between nine effectors that were tested, two only (Raxil and Simasol) proved to have the highest degree of efficacy (up to 100%), Hattric and pine oil were the most effective treatments (98.4 and 97.9%, respectively). Gamma radiation at 250 Gy followed by clove oil and castor oil changed reactions to moderately resistant against U. nuda. Infection of barley seed caused significant differences among all studied plant traits.

Fusarium isolates from Belgium causing wilt in lettuce show genetic and pathogenic diversity

AbstractFusarium oxysporum f. sp. lactucae race 4 causes vascular necrosis and wilting of lettuce. First observed in Belgium in 2015, the lack of disease resistance in commercial cultivars allowed this pathogen to spread to nearly the entire Belgian production area within 4 years. Different levels of disease development were observed in different commercial greenhouses. To help explain this variation, we collected 78 Fusarium isolates and characterized them both physiologically and genetically. Molecular race identification indicated that 91% of the isolates belonged to race 4, while 6% of the isolates belonged to race 1, which was not previously reported in Belgium. Pathogenicity assays using differential cultivars confirmed the molecular race assignment of selected isolates. Cultivar Patriot was identified as a suitable new differential cultivar to race 4. Race 4 isolates were more aggressive than race 1 isolates at 24°C, but only when using chlamydospore inoculum instead of a root dip assay containing microconidia. Variation in pathogenicity and aggressiveness of the races may explain differences in disease development in commercial greenhouses. Based on genotyping‐by‐sequencing (GBS), race 1 and race 4 isolates were highly similar to reference isolates. Fusarium curvatum, F. oxysporum f. sp. tulipae and F. oxysporum f. sp. rhois were phylogenetically separated from F. oxysporum f. sp. lactucae races 1 and 4 based on the GBS data, but not when using multilocus sequence data. Within F. oxysporum f. sp. lactucae race 4, the GBS data differentiated two rather homogeneous groups, suggesting at least two introductions. However, the two groups did not differ in aggressiveness.