A2 Mating Type Research Articles

Studies on the Presence of Different Mating Types of<em> Phytophthora infestans</em> in Nuwara Eliya Sri Lanka

Late blight, caused by Phytophthora infestans is one of the most devastating diseases in potato cultivation. It results in direct crop losses but also causes a considerable amount of cost for disease management. The primary objective of this study was to investigate P. infestans mating types in selected areas in Sri Lanka and evaluate the risks associated with mating types. In this experiment potato leaves from a range of late blight severity levels were sampled from infected farmer fields. The pathogen was isolated by leaf culture method and confirmed by microscopic observations. The mating types of each isolate were studied by a Metalaxyl sensitivity test. Mating type A1 has been previously reported in Sri Lanka, but mating type A2 was not reported since 2013. Results of this study reveal that mating type A1 was more prominent than A2 (75% and 50% respectively). A2 was reported only from two fields. These observations prove the presence and emergence of mating type A2 in Sri Lankan potato fields. Gradual increase of detectable A2 mating types suggests a high possibility of pathogen inoculum movement between the locations over time. The presence of both mating types raises the possibility of sexual reproduction and generation of oospores. The ratio of A1: A2: (A1+A2) in the current study was 2:1:1. This could permit genetic recombination with the biological evolution of fungicideresistant types of P. infestans species. This study emphasizes the importance of investigations on the mating types to manage late blight disease in Sri Lankan potato fields.

Survey of oomycetes and pathogenicity of Phytophthora cinnamomi associated with root rot disease of western white pine (Pinus monticola)

Western white pine (Pinus monticola) with genetic tolerance to white pine blister rust disease caused by Cronartium ribicola constitutes an invaluable resource for the mitigation of this disease as well as the restoration of the species and its utilization in managed forests. In the last several years, typical root-rot symptoms and sudden tree death have been observed in some western white pine plantations located in British Columbia (BC) in Canada, and a few Phytophthora species were found to be associated with symptomatic trees. Our objectives were to survey one of the plantations located in south-west BC on Vancouver Island to confirm the presence of these Phytophthora species and other oomycetes and test their pathogenicity on western white pine by completing Koch’s postulates. We used direct plating of root samples on PARP(H)-CMA media and by baiting for oomycetes in soil and root samples. Ten oomycetes, including four Phytophthora species (Phytophthora cinnamomi, P. cactorum-like, P. cryptogea and P. pseudocryptogea) were isolated from roots and soil samples of diseased Pi. monticola trees. The Phytophthora species were identified using ITS, β-tubulin and TEF1 gene regions. Koch’s postulates were validated with controlled inoculations of 1-year old seedlings; mortality occurred as early as 15 days after inoculation with P. cinnamomi when the seedlings were maintained in warm temperature conditions (25°C). Molecular typing of two P. cinnamomi cultures using Oxford Nanopore MinIONTM sequencing indicated that they belong to one of the two main clonal clusters of mating type A2 that have been recognized as the main drivers of the global P. cinnamomi epidemic.

First Report of Plumed Cockscomb (Celosia argentea var. plumosa) Stem Blight Caused by Phytophthora nicotianae in Taiwan.

Celosia spp. is a widely cultivated ornamental plant in gardens or parks in Taiwan. In September 2021, withering leaves and grayish-brown lesions were observed on the lower stem of plumed cockscombs (C. argentea var. plumosa) in Taichung City, with an incidence of about 22% in 136 plants after continuous precipitation, impacting the aesthetic value of the landscape. Symptomatic plants were collected, surface disinfected with 70% EtOH for ~20 sec., blotted dried, and excised diseased tissues (~ 3×3 mm2) were placed on 2% water agar. Four representative isolates were obtained after purification and the colonies were white with aerial and non-septated hyphae on V8 agar for 7 days. Sporangia were ovoid, ellipsoid or obpyriform, papillate, (26.3-55.9) 38.0 × 29.0 (20.1-40.6) µm (n = 200) (Ahonsi et al. 2007). Chlamydospores were spherical, terminal or intercalary, 26.0 (15.1-40.4) µm (n = 200). All isolates belong to A2 mating type with amphigynous antheridia and plerotic oospores, 21.0 (17.7-25.7) µm (n = 200), resembling the descriptions of Phytophthora (Erwin & Ribeiro 1996). For molecular identification, sequences of the ITS, β-tubulin (β-tub), and EF-1α regions of all isolates were amplified using ITS1/ITS4, TUBUF2/TUBUR1, and ELONGF1/ELONGR1 primers, respectively (White et al. 1990; Kroon et al. 2004). BLAST analyses of isolates cap1-2 (ITS: OQ581785; β-tub: OQ590022; EF-1α: OQ590026), cap1-3 (ITS: OQ581786; β-tub: OQ590023; EF-1α: OQ590027), cap2-1 (ITS: OQ581787; β-tub: OQ590024; EF-1α: OQ590028), and cap2-2 (ITS: OQ581788; β-tub: OQ590025; EF-1α: OQ590029) showed 100% of ITS identity, 99.5 to 99.9% of β-tub identity, and 99.4 to 99.6% of EF-1α identity with Phytophthora nicotianae (ITS: MG865551; β-tub: MH493987; EF-1α: MH359043). Phylogenetic trees were constructed using concatenated ITS, β-tub, and EF-1α sequences based on maximum likelihood with a GTR+G model in MEGA X and Bayesian inference method in Geneious Prime 2022.2. All isolates were clustered in P. nicotianae with similar topology, thereby were identified as P. nicotianae. To confirm pathogenicity, 7 to 10-day-old seedlings and 6-week-old plumed cockscomb plants were inoculated in separate trials and each experiment was conducted twice. For each seedling, the lower stem was inoculated with 50 μl of zoospore suspension (104 zoospores/ml), 3 plants per isolate, and then incubated at 30±2℃ with 12 h light. For adult plants, each was inoculated with mycelial plugs from one V8 plate of 10-day-old P. nicotianae, 5 plants per isolate, and incubated at 25±2℃ in a greenhouse. Control plants were inoculated with sterile water and V8 agar plugs, respectively. Stem and root rot were observed on seedlings 4 days after inoculation while wilting and lower stem browning were observed on adult plants 2 months after inoculation. All control plants remained healthy at the end of repeated trials and identical pathogens were re-isolated only from symptomatic plants, thus fulfilling Koch's rules. P. nicotianae has been reported causing root rot and stem necrosis not only on cockscomb (C. plumosa Hort. ex Burvenich) in Argentina (Frezzi 1950), but also infecting several ornamental plants recently in Taiwan (Ann et al. 2018). To our knowledge, this is the first report of stem blight caused by P. nicotianae on plumed cockscombs in Taiwan. This finding suggests limited options for landscaping and the host preference of the isolates obtained in this study should warrant further studies.

Insights into the population dynamics of phytophthora species associated with arecanut fruit rot disease

Phytophthora, a ubiquitous filamentous oomycete, causes huge yield losses and is fatal to arecanut palms in case of severe infection. From 2014 onwards, a severe fruit rot epidemic has been recorded during the South-West monsoon season in major arecanut-growing states of South India. We have assessed the diversity and delineated the population structure of Phytophthora isolates infecting arecanut in India using a combination of morphological traits, multi-gene profiling, haplotype analysis, and pathogenicity. Ninety-eight Phytophthora isolates were obtained from infected samples collected from disease-endemic regions of Karnataka, Kerala, and Goa states in southern India from 2014 to 2019. Morphological traits coupled with phylogenetic analysis using the loci ITS, β-tub, TEF-1α, and Cox-II identified the A2 mating type of P. meadii as the predominant species together with two isolates of homothallic P. heveae. Linkage disequilibrium analysis revealed significant diversity in the form of single nucleotide polymorphisms (SNPs) from the concatenated sequence dataset. Population structure analysis using 590 SNPs demonstrated the existence of four population groups (sub-populations at K = 4 and K = 2) and significant diversity in the geo-distant Phytophthora populations. In summary, 22 haplotypes were identified from the representative 40 isolates with higher haplotype diversity of 1.23 and relatively varying haplotype frequency. Pathogenicity assays confirmed that both the species of Phytophthora were pathogenic to arecanut, while P. meadii coastal region isolates exhibited more virulence compared to others.

Machine Learning-Based Identification of Mating Type and Metalaxyl Response in Phytophthora infestans Using SSR Markers.

Phytophthora infestans is the causal agent of late blight in potato. The occurrence of P. infestans with both A1 and A2 mating types in the field may result in sexual reproduction and the generation of recombinant strains. Such strains with new combinations of traits can be highly aggressive, resistant to fungicides, and can make the disease difficult to control in the field. Metalaxyl-resistant isolates are now more prevalent in potato fields. Understanding the genetic structure and rapid identification of mating types and metalaxyl response of P. infestans in the field is a prerequisite for effective late blight disease monitoring and management. Molecular and phenotypic assays involving molecular and phenotypic markers such as mating types and metalaxyl response are typically conducted separately in the studies of the genotypic and phenotypic diversity of P. infestans. As a result, there is a pressing need to reduce the experimental workload and more efficiently assess the aggressiveness of different strains. We think that employing genetic markers to not only estimate genotypic diversity but also to identify the mating type and fungicide response using machine learning techniques can guide and speed up the decision-making process in late blight disease management, especially when the mating type and metalaxyl resistance data are not available. This technique can also be applied to determine these phenotypic traits for dead isolates. In this study, over 600 P. infestans isolates from different populations-Estonia, Pskov region, and Poland-were classified for mating types and metalaxyl response using machine learning techniques based on simple sequence repeat (SSR) markers. For both traits, random forest and the support vector machine demonstrated good accuracy of over 70%, compared to the decision tree and artificial neural network models whose accuracy was lower. There were also associations (p < 0.05) between the traits and some of the alleles detected, but machine learning prediction techniques based on multilocus SSR genotypes offered better prediction accuracy.

First report of Phytophthora infestans genotype US17 causing late blight on potato and tomato in British Columbia, Canada

Potato and tomato are grown in all Canadian provinces and contribute billions of dollars in exports and sales. Late blight, caused by Phytophthora infestans, is a destructive disease of potato and tomato worldwide and is responsible for millions of dollars in losses annually. Changes in pathogen strains were reported to be associated with late blight epidemics in several regions and pose challenges to late blight management. In British Columbia (BC), diverse strains of P. infestans were reported. Using multiplex microsatellite or simple sequence repeat (SSR) markers, we identified the new genotype US17 of P. infestans from samples of potato and tomato with late blight in BC. All isolates were of the A1 mating type. One, five, and one isolates were sensitive, moderately resistant, and highly resistant to metalaxyl-m, respectively. This is the first report of the occurrence of the US17 genotype in BC and Canada. The US17 strain was also able to cause tuber rot and foliar blight symptoms in potato in pathogen-inoculated assays.

Report of Phytophthora capsici (mating type A1) isolates associated with crown‐rot disease of Capsicum annuum in Guatemala

AbstractWilting and crown‐rot symptoms were observed in sweet pepper (Capsicum annuum) plants in field surveys carried out in 2021 across major vegetable‐producing areas of three municipalities of Guatemala. Phytophthora capsici (Peronosporales, Peronosporaceae) isolates were identified in association with these symptoms based upon morphological and phylogenetic analyses using nucleotide sequence information of the Cox2 mitochondrial DNA (mtDNA) region. Biological tests confirmed P. capsici mating type A1 isolates as the causal agents of the disease. Symptoms similar to those observed in the field were obtained after controlled inoculation of C. annuum ‘Tico’ seedlings. Solanum habrochaites ‘LA 404’ and C. annuum ‘Criollo de Morelos’ displayed strong resistance reaction against all Guatemalan isolates. Even though P. capsici is a cosmopolitan pathogen, this is the first molecular confirmation of this pathogen infecting C. annuum in Guatemala.

Understanding the genotypic and phenotypic structure and impact of climate on Phytophthora nicotianae outbreaks on potato and tomato in the eastern US.

Samples from potato fields with late blight-like symptoms were collected from eastern North Carolina in 2017 and the causal agent was identified as Phytophthora nicotianae. We have identified P. nicotianae in potato and tomato from North Carolina, Virginia, Maryland, Pennsylvania, and New York. Ninety-two field samples were collected from 46 fields and characterized for mefenoxam sensitivity, mating type, and SSR genotype using microsatellites. Thirty two percent of isolates were the A1 mating type, while 53% were A2 mating type. In six cases, both A1 and A2 mating type were detected in the same field in the same year. All isolates tested were sensitive to mefenoxam. Two genetic groups were discerned based on STRUCTURE analysis: one included samples from North Carolina and Maryland, and one included samples from all five states. The data suggest two different sources of inoculum from the field sites sampled. Multiple haplotypes within a field and the detection of both mating types in close proximity suggests that P. nicotianae may be reproducing sexually in North Carolina. There was a decrease in the average number of days with weather suitable for late blight, from 2012-2016 to 2017-2021 in all of the NC counties where P. nicotianae was reported. Phytophthora nicotianae is more thermotolerant than P. infestans and grows at higher temperatures (25-35°C) than P. infestans (18-22°C). Late blight outbreaks have decreased in recent years and first reports of disease are later, suggesting that the thermotolerant P. nicotianae may cause more disease as temperatures rise due to climate change.

Genotypic characterization of Phytophthora infestans populations in Bangladesh

AbstractLate blight, caused by Phytophthora infestans, is an economically important disease of potato that causes significant yield losses, with severe outbreaks regularly occurring in Bangladesh. The objective of this study was to do a large‐scale survey of potato fields in the main potato‐growing divisions of Bangladesh examining genotypic diversity of P. infestans populations. A total of 160 samples were collected in 2018 from both potato (n = 140) and tomato (n = 20). Isolates were mainly collected on FTA cards (n = 143), but 17 were also collected and isolated into pure culture. Microsatellite analysis revealed high levels of subclonal diversity in P. infestans populations with 116 multilocus genotypes recorded from 160 samples. Comparisons with standards of European and US isolates showed that 74% of samples could be categorized as genotype EU_13_A2, 7% clustered near EU_6_A1 and EU_1_A1, and 19% were unique. Discriminant analysis of principal components showed that the P. infestans population clustered into four distinct groups: a main group that contained most of the samples from potato, two distinct tomato groups and one group of samples originating from the division of Mymensingh. Of 17 isolates from cultures, 16 were EU_13_A2 and one was EU_6_A1; 15 were insensitive to metalaxyl‐M. Out of 160 samples, 158 were categorized as mating type A2 and two as mating type A1. These results indicate that Bangladesh populations of P. infestans from potato, like those from neighbouring countries, are dominated by genotype EU_13_A2. However, populations from tomato were distinct and appear to be specific to tomato.

QTL mapping of resistance to Pseudoperonospora cubensis clade 2, mating type A1, in Cucumis melo and dual-clade marker development.

This is the first identification of QTLs underlying resistance in Cucumis melo to an isolate of Pseudoperonospora cubensis identified as Clade 2/mating type A1. Pseudoperonospora cubensis, causal organism of cucurbit downy mildew (CDM), causes severe necrosis and defoliation on Cucumis melo (melon). A recombinant inbred line population (N = 169) was screened against an isolate of P. cubensis (Clade 2/mating type A1) in replicated greenhouse and growth chamber experiments. SNPs (n = 5633 bins) identified in the RIL population were used for quantitative trait loci (QTL) mapping. A single major QTL on chromosome 10 (qPcub-10.3-10.4) was consistently associated with resistance across all experiments, while a second major QTL on chromosome 8 (qPcub-8.3) was identified only in greenhouse experiments. These two major QTLs were identified on the same chromosomes (8 and 10) but in different locations as two major QTLs (qPcub-8.2 and qPcub-10.1) previously identified for resistance to P. cubensis Clade 1/mating type A2. Kompetitive allele-specific PCR (KASP) markers were developed for these four major QTLs and validated in the RIL population through QTL mapping. These markers will provide melon breeders a high-throughput genotyping toolkit for development of melon cultivars with broad tolerance to CDM.

Population Dynamics of Phytophthora infestans in Egypt Reveals Clonal Dominance of 23_A1 and Displacement of 13_A2 Clonal Lineage.

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt.

Virus-Induced Silencing of a Sequence Coding for Loricrin-like Protein in Phytophthora infestans upon Infection of a Recombinant Vector Based on Tobacco Mosaic Virus

Phytophthora infestans is the oomycete responsible for late blight disease of Solanaceae that causes both yield and economic losses. With the aim of reducing plant wilt and high management costs mainly due to wide fungicide applications, alternative eco-sustainable control strategies are needed. RNA interference (RNAi) is a powerful tool for gene function studies that can be accomplished by constitutive transformation or transient expression such as virus-induced gene silencing (VIGS) experiments. VIGS makes use of viruses to deliver sequences homologous to a target gene fragment and trigger RNAi. Indeed, a P. infestans ortholog of plant loricrin-like protein (LLP), named PiLLP, has been silenced using the direct infection of a recombinant vector based on the plant virus tobacco mosaic virus (TMV-PiLLP-1056), aiming to reduce the oomycete sexual reproduction. For this purpose, the gene coding for the green fluorescent protein (GFP) present in the TMV-GFP-1056 vector has been replaced with an antisense construct obtained by fusion PCR of the PiLLP 5′-UTR and 3′-UTR sequences. Here, we show that RNAi can be expressed in the A1 mating type of P. infestans strain 96.9.5.1 by VIGS using the direct infection of TMV-PiLLP-1056. We provide evidence that the recombinant vector can enter, replicate, and persist in mycelia of P. infestans where it induces the partial downregulation of the PiLLP transcript. Compared with the wild-type, the PiLLP-silenced A1 mating type had slower colony growth and a diminished virulence in detached tomato leaflets. This seems to be the first evidence of a constitutive gene downregulation of P. infestans using a recombinant vector based on a plus-sense RNA plant virus.

Phytophthora × cambivora as a Major Factor Inciting the Decline of European Beech in a Stand within the Southernmost Limit of Its Natural Range in Europe.

The objective of this study was to investigate the role of the oomycete Phytophthora × cambivora in the decline affecting European beech (Fagus sylvatica) in the Nebrodi Regional Park (Sicily, southern Italy). In a survey of a beech forest stand in the heart of the park, Phytophthora × cambivora was the sole Phytophthora species recovered from the rhizosphere soil and fine roots of trees. Both A1 and A2 mating type isolates were found. Direct isolation from the stem bark of trees showing severe decline symptoms and bleeding stem cankers yielded exclusively P. gonapodyides, usually considered as an opportunistic pathogen. The mean inoculum density of P. × cambivora in the rhizosphere soil, as determined using the soil dilution plating method and expressed in terms of colony forming units (cfus) per gm of soil, the isolation frequency using leaf baiting, and the percentage of infected fibrous roots from 20 randomly selected beech trees with severe decline symptoms (50 to 100 foliage transparency classes) were 31.7 cfus, 80%, and 48.6%, respectively. These were significantly higher than the corresponding mean values of 20 asymptomatic or slightly declining trees, suggesting P. × cambivora is a major factor responsible for the decline in the surveyed stand.

Phenotypic and Genotypic Analysis of the Population of Phytophthora infestans in Bangladesh Between 2014 and 2019

In Bangladesh, the third largest producer of potatoes in Asia, late blight, caused by Phytophthora infestans, is the major constraint to production. Nevertheless, there is a lack of published information on the pathogen population. A collection of 69 isolates obtained from samples of infected potato foliage collected in 2018–2019 varied significantly in their aggressiveness to detached potato leaflets and tuber slices of cv. Diamant. On leaflets, most isolates were highly aggressive, colonizing more than half the surface in 7 days. On tuber slices, while some isolates were highly aggressive, those collected in 2019 were less so. There was a significant effect of the locations from which the isolates were obtained on aggressiveness to both leaflets and tuber slices, but aggressiveness to leaflets and to tuber slices was not significantly correlated. All isolates were A2 mating type and either intermediate (38%) or resistant (62%) in sensitivity to the fungicide metalaxyl. A subset of 24 isolates all had mitochondrial DNA haplotype Ia.Samples of P. infestans DNA (124) from infected potato foliage obtained from Bangladesh (2014–2019) were genotyped by 12-plex SSR. The vast majority (95%) were assigned to EU_13_A2, which was present in every year in which samples were collected. Numerous EU_13_A2 variants were identified, many specific to Bangladesh. Six samples (from 2017 to 2019) had genotypes distinct from EU_13_A2 and with no close match to known European lineages. It is concluded that the current P. infestans population of Bangladesh is dominated by the aggressive lineage EU_13_A2, also dominant in India and Pakistan. The implications of this for control of late blight are discussed.

Genotypic and Phenotypic Structure of the Population of Phytophthora infestans in Egypt Revealed the Presence of European Genotypes.

Late blight disease of potato and tomato, caused by Phytophthora infestans, results in serious losses to Egyptian and global potato and tomato production. To understand the structure and dynamics of the Egyptian population of P. infestans, 205 isolates were collected from potato and tomato plants during three growing seasons in 2010–2012. The characterization was achieved by mating-type assay, metalaxyl sensitivity assay, and virulence pattern. Additionally, genotyping of 85 Egyptian isolates and 15 reference UK isolates was performed using 12 highly informative microsatellite (SSR) markers David E. L. Cooke and five effector (RxLR) genes. Mating-type testing showed that 58% (118 of 205) of the isolates belonged to mating type A1, 35% (71 isolates) to mating type A2, and the rest 8% (16 isolates) were self-fertile. The phenotype of metalaxyl response was represented as 45% resistant, 43% sensitive, and 12% as intermediate. Structure analysis grouped the 85 identified genotypes into two main clonal lineages. The first clonal lineage comprised 21 isolates belonging to A2 mating type and 8 self-fertile isolates. This clonal lineage was identified as Blue_13 or EU_13_A2. The second main clonal lineage comprised 55 isolates and was identified as EU_23_A1. A single isolate with a novel SSR genotype that formed a distinct genetic grouping was also identified. The effector sequencing showed good correspondence with the virulence data and highlighted differences in the presence and absence of loci as well as nucleotide polymorphism that affect gene function. This study indicated a changing population of P. infestans in Egypt and discusses the findings in the context of late blight management.

Metabolomic and Physiological Changes in Fagus sylvatica Seedlings Infected with Phytophthora plurivora and the A1 and A2 Mating Types of P. ×cambivora

Phytophthora infections are followed by histological alterations, physiological and metabolomic adjustments in the host but very few studies contemplate these changes simultaneously. Fagus sylvatica seedlings were inoculated with A1 and A2 mating types of the heterothallic P. ×cambivora and with the homothallic P. plurivora to identify plant physiological and metabolomic changes accompanying microscope observations of the colonization process one, two and three weeks after inoculation. Phytophthora plurivora-infected plants died at a faster pace than those inoculated with P. ×cambivora and showed higher mortality than P. ×cambivora A1-infected plants. Phytophthora ×cambivora A1 and A2 caused similar progression and total rate of mortality. Most differences in the physiological parameters between inoculated and non-inoculated plants were detected two weeks after inoculation. Alterations in primary and secondary metabolites in roots and leaves were demonstrated for all the inoculated plants two and three weeks after inoculation. The results indicate that P. plurivora is more aggressive to Fagus sylvatica seedlings than both mating types of P. ×cambivora while P. ×cambivora A1 showed a slower infection mode than P. ×cambivora A2 and led to minor plant metabolomic adjustments.

Characterization and fungicides sensitivity of Phytophthora cinnamomi isolates causing avocado root rot in Zitácuaro, Michoacán

&lt;em&gt;Phytophthora cinnamomi&lt;/em&gt; is the pathogen most frequently associated with avocado root rot. In Zitácuaro, Michoacán, production has increased by 19.8%; however, there are no studies of root rot in this area. The objective of the study was to characterize the isolates obtained from avocado roots and assess the sensitivity to fungicides. Samples from 5 avocado orchards were collected, sampling 5 trees per orchard (a total of 25 samples). The samples isolated were characterized morphological and molecularly. Mating type was analyzed using reference isolates of&lt;em&gt; P. cinnamomi&lt;/em&gt; A1 (isolate from camelia) and A2 (isolate from avocado). To confirm the pathogenicity, tests were performed on avocado fruits with the isolates. The sensitivity of 15 isolates to potassium phosphite and to metalaxyl-M at different concentrations was evaluated&lt;em&gt; in vitro&lt;/em&gt;. In a subgroup of six isolates, it was evaluated whether there was a relationship between growth rate and potassium phosphite sensitivity. Fifteen isolates were obtained with coenocytic coraloid mycelium, chlamydospores, sporangia without papilla, ovoid to ellipsoid, with internal proliferation, heterothallic with mating type A2, with amphigynous antheridia and plerotic oospores, characteristics consistent with &lt;em&gt;P. cinnamomi&lt;/em&gt;. The inoculated isolates were pathogenic on avocado fruits. The isolates were more sensitive to potassium phosphite than to metalaxyl-M, with mean EC50 values of 24.62 and 0.215 ?g mL-1 of i.a., respectively. No relationship was observed between growth rate and potassium phosphite sensitivity. It is necessary to obtain a greater number of&lt;em&gt; P. cinnamomi&lt;/em&gt; isolates for virulence studies.

First Report of Stem and Foliage Blight of Chrysanthemum Caused by Phytophthora drechsleri in the United States.

Chrysanthemum (Chrysanthemum × morifolium) plants exhibiting stem and foliage blight were observed in a commercial nursery in eastern Oklahoma in June 2019. Disease symptoms were observed on ~10% of plants during a period of frequent rain and high temperatures (26-36°C). Dark brown lesions girdled the stems of symptomatic plants and leaves were wilted and necrotic. The crown and roots were asymptomatic and not discolored. A species of Phytophthora was consistently isolated from the stems of diseased plants on selective V8 agar (Lamour and Hausbeck 2000). The Phytophthora sp. produced ellipsoid to obpyriform sporangia that were non-papillate and persistent on V8 agar plugs submerged in distilled water for 8 h. Sporangia formed on long sporangiophores and measured 50.5 (45-60) × 29.8 (25-35) µm. Oospores and chlamydospores were not formed by individual isolates. Mycelium growth was present at 35°C. Isolates were tentatively identified as P. drechsleri using morphological characteristics and growth at 35°C (Erwin and Ribeiro 1996). DNA was extracted from mycelium of four isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS 4 and ITS 6. The PCR product was sequenced and a BLASTn search showed 100% sequence similarity to P. drechsleri (GenBank Accession Nos. KJ755118 and GU111625), a common species of Phytophthora that has been observed on ornamental and vegetable crops in the U.S. (Erwin and Ribeiro 1996). The gene sequences for each isolate were deposited in GenBank (accession Nos. MW315961, MW315962, MW315963, and MW315964). These four isolates were paired with known A1 and A2 isolates on super clarified V8 agar (Jeffers 2015), and all four were mating type A1. They also were sensitive to the fungicide mefenoxam at 100 ppm (Olson et al. 2013). To confirm pathogenicity, 4-week-old 'Brandi Burgundy' chrysanthemum plants were grown in 10-cm pots containing a peat potting medium. Plants (n = 7) were atomized with 1 ml of zoospore suspension containing 5 × 103 zoospores of each isolate. Control plants received sterile water. Plants were maintained at 100% RH for 24 h and then placed in a protected shade-structure where temperatures ranged from 19-32°C. All plants displayed symptoms of stem and foliage blight in 2-3 days. Symptoms that developed on infected plants were similar to those observed in the nursery. Several inoculated plants died, but stem blight, dieback, and foliar wilt were primarily observed. Disease severity averaged 50-60% on inoculated plants 15 days after inoculation. Control plants did not develop symptoms. The pathogen was consistently isolated from stems of symptomatic plants and verified as P. drechsleri based on morphology. The pathogenicity test was repeated with similar results. P. drechsleri has a broad host range (Erwin and Ribeiro 1996; Farr et al. 2021), including green beans (Phaseolus vulgaris), which are susceptible to seedling blight and pod rot in eastern Oklahoma. Previously, P. drechsleri has been reported on chrysanthemums in Argentina (Frezzi 1950), Pennsylvania (Molnar et al. 2020), and South Carolina (Camacho 2009). Chrysanthemums are widely grown in nurseries in the Midwest and other regions of the USA for local and national markets. This is the first report of P. drechsleri causing stem and foliage blight on chrysanthemum species in the United States. Identifying sources of primary inoculum may be necessary to limit economic loss from P. drechsleri.

Phytophthora heterospora sp. nov., a New Pseudoconidia-Producing Sister Species of P. palmivora.

Since 1999, an unusual Phytophthora species has repeatedly been found associated with stem lesions and root and collar rot on young olive trees in Southern Italy. In all cases, this species was obtained from recently established commercial plantations or from nursery plants. Morphologically, the Phytophthora isolates were characterized by the abundant production of caducous non-papillate conidia-like sporangia (pseudoconidia) and caducous papillate sporangia with a short pedicel, resembling P. palmivora var. heterocystica. Additional isolates with similar features were obtained from nursery plants of Ziziphus spina-christi in Iran, Juniperus oxycedrus and Capparis spinosa in Italy, and mature trees in commercial farms of Durio zibethinus in Vietnam. In this study, morphology, breeding system and growth characteristics of these Phytophthora isolates with peculiar features were examined, and combined mitochondrial and nuclear multigene phylogenetic analyses were performed. The proportion between pseudoconidia and sporangia varied amongst isolates and depended on the availability of free water. Oogonia with amphigynous antheridia and aplerotic oospores were produced in dual cultures with an A2 mating type strain of P. palmivora, indicating all isolates were A1 mating type. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora; thus, they constitute a separate taxon. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in stem inoculation tests.

Identification of biosynthetic intermediates for the mating hormone α2 of the plant pathogen Phytophthora.

The heterothallic group of the plant pathogen Phytophthora can sexually reproduce between the cross-compatible mating types A1 and A2. The mating hormone α2, produced by A2 mating type and utilized to promote the sexual reproduction of the partner A1 type, is known to be biosynthesized from phytol. In this study, we identified 2 biosynthetic intermediates, 11- and 16-hydroxyphytols (1 and 2), for α2 by administering the synthetic intermediates to an A2-type strain to produce α2 and by administering phytol to A2 strains to detect the intermediates in the mycelia. The results suggest that α2 is biosynthesized by possibly 2 cytochrome P450 oxygenases via 2 hydroxyphytol intermediates (1 and 2) in A2 hyphae and secreted outside.