Mating type revulsion and dynamic genetic diversity of Phytophthora infestans during 2010–2021 in yunnan, China

doi:10.21203/rs.3.rs-3134005/v1

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Mating type revulsion and dynamic genetic diversity of Phytophthora infestans during 2010–2021 in yunnan, China

https://doi.org/10.21203/rs.3.rs-3134005/v1

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Potato late blight caused by Phytophthora infestans is the most severe problem in the Yunnan Province. To understand the population dynamics of P. infestans in Yunnan during 2010–2020, 357 isolates were collected from Dali and Kunming, purified, and analyzed for the mating type, mitochondrial DNA (mtDNA) haplotype, metalaxyl resistance, pathotype, and simple sequence repeat (SSR) DNA fingerprints. The A1 mating type was dominant before 2012, but since 2012, the A2, A1A2 and self-fertile types became dominant and fewer A1 isolates were obtained over the years. Among the 357 isolates, only two were IIa mtDNA haplotypes and the rest were Ia mtDNA haplotypes. Pathogenicity assays using R1-R11 gene carried hosts revealed 67 pathotypes among all test isolates. SSR analysis identified 242 multi locus genotypes(MLGs), combined principal component analysis indicated that populations in 2010, 2011 and 2012 predominantly adopted asexual life cycles, but from 2013 to 2021, limited sexual reproduction likely occurred. UPGMA analysis indicated that five test isolates collected from Dali and Kunming from 2013 to 2015 but not in other years represented the EU_13_A2 lineage. Our results support the hypothesis that clonal reproduction, combined with host selection and limited sexual reproduction, underlies the dynamic genetic diversity of P. infestans in Yunnan.

Genotype

potato late blight

Phytophthora infestans

population structure

sexual reproduction.

In sexual oomycete species, mating systems are often considered in terms of the degree of inbreeding that occasionally occurs in populations (1). Oomycetes that undergo asexual reproduction in the field often tend to be genetically conserved, leading to lower genotypic diversity within the population. In contrast, sexual reproduction, which involves the fusion of two or more mating types to produce the offspring, may increase the possibility of genotypic and phenotypic variation (2).

Mating systems of several fungal phytopathogens have been studied. For instance, the heterothallic diploid oomycete Phytophthora infestans, the pathogen responsible for the Irish famine during 1843–1845, typically has two mating systems, namely A1 and A2. The hormones produced by the A1 and A2 mating types stimulate the production of oogonia and antheridia, respectively (3). Oospores produced from a mating event, coming into contact with both A1 and A2 typical strains and serving as the inoculum source in the soil, contribute to overwintering survival, initial infection process, and population genetic variation. Meanwhile, in some strains, self-fertile and A1A2 (can pair with both A1 and A2 strains, but not with self-fertile strains, to produce oospores) mating types may occasionally exist in the field (4). Furthermore, when both mating types coexist at a location, a sexually reproducing population can produce numerous and diverse genotypes, rendering disease control extremely difficult. For instance, the ability of a fungal pathogen to conquer the novel host resistance (R) genes can lead to the development of fungicide resistance or increase in pathogen virulence. To facilitate sexual reproduction, both mating types must be present in the same field, or infect the same leaf, and produce abundant oospores. Prior to the 1980s, the A2 mating types were restricted to Mexico, and sexual reproduction was documented only in this region (5). In the early 1980s, the A2 mating type was first detected in Europe (6) and subsequently spread to many other countries (3). The pathogenic populations in eastern Europe and North America have undergone a major transition, with the pre-1980 population being replaced by new clonal lineages, due to occasional shifts in mating systems or sexual recombination; for example, in the United States, the dominant P. infestans population has shifted from the former US-1 (mating type A1, mtDNA haplotype Ib) to the novel US-8 (mating type A2, mtDNA haplotype Ib). In 2004, the emergence of a highly virulent and metalaxyl-resistant A2 mating type strain of P. infestans, Blue_13, was first reported in the Netherlands (7), representing a major threat to potato cultivation in western and eastern Europe (8, 9), India (10), China (7), Russia (11), Italy and Algeria (12). In Europe, the genotype still sampled but a continual declining proportion less than 5% compare to 35% in 2014 (EuroBlight population maps). Tracking such population changes requires an array of methods, as reviewed by Cooke and Lees (13).

The use of marker systems has simplified the study of the occurrence of sexual reproduction in P. infestans. Genotypic diversity resulting from mating type shifts or sexual reproduction is often measured using various markers. Genotypic DNA fingerprinting, which involves diploid chromosome alleles, such as isozymes (14), amplified fragment length polymorphisms (15), RG57 fingerprint patterns (16), restriction fragment length polymorphisms, simple sequence repeat (SSR) microsatellites, or sequences within or among populations, as well as mitochondrial haploid diversity, has been used as a routine protocol (17). In addition, phenotypes have been assessed based on mating type, metalaxyl-resistance, and pathogenicity. Currently, capillary electrophoresis-based SSR screening is the favored approach. It is a potent and highly stable tool to analyze asexual clonal lineages as well as sexual lineages with high genotype diversity. This method can be used to examine the presence and significance of population changes in the field and gain valuable information for managing potato late blight (PLB) epidemics and developing control strategies.

Yunnan is a southwestern province in China, covering 94% of the mountainous areas. In this province, the history of potato cultivation dates as early as the middle of the Qing dynasty in 1731 (18). Potatoes are a major food crop in Yunnan, grown into autumn (July to December), winter (October to March), spring potatoes (March to July), and early spring potato (December to May) production zones (19). However, PLB caused by P. infestans is one of the most severe problems during cultivation in major production zones. In 1999, A2 mating type isolates of P. infestans were detected, albeit few, in Yunnan for the first time (20). Subsequently, oospores were also identified in the field (21), and after 2008, A2 and self-fertile isolates became dominant populations in most potato-producing areas. The 13_A2 Blue genotype has also been detected in this province (7). Such changes may greatly impact the population genetic dynamics, virulence, and fungicide resistance of potato pathogens.

To this end, for better understanding the population genetic diversity of P. infestans in Yunnan and infer potential PLB epidemic trends, we collected pathogens for last 12 years from two major production regions in the province and performed molecular SSR marker-based and phenotypic analyses. The main objectives of the present study were (i) to estimate the genotypic and phenotypic variation and genetic dynamics of P. infestans in Dali and Kunming in Yunnan over 12 years; (ii) to investigate the possibility of sexual reproduction among populations; and (iii) to explore the possible involvement of the A2 and self-fertile mating type strains of P. infestans following genetic drift around 2012 in recent destructive late blight epidemics in the province.

Strain collection, isolation, and culture conditions

A total of 357 strains collected from Dali and Kunming in Yunnan from 2010 to 2021 were included in the present study (Table S1; Fig. 1). Samples were collected from villages separated by at least 1 km; infected leaves were placed in 9 cm sterile petri dishes containing a 5-mm-thick layer of 2% water-agar medium at 18°C for 48h. To purify P. infestans, a single filamentous colony was transferred to rye agar B (22) supplemented with ampicillin (50 mg/l), rifampin (15 mg/l), and pentachloronitro benzene (PCNB, 5 mg/l) and maintained at 18℃ for five to seven days. Then, a single piece of mycelium was cut and plated onto fresh rye agar B for additional seven days. Only one clone was selected from one infected sample. All isolates were identified as P. infestans through internal transcribed spacer (ITS) sequencing and homology analysis using the NCBI database. Strain 3928A (genotype 13_A2) was obtained from Dr. Zhengdong Tian (Huazhong Agricultural University, China). Reference isolates 88069 and 88133 for mating type identification were obtained from Dr. Liyun Guo (China Agriculture University).

DNA extraction

Mycelia (5 mg) were obtained from 14-day-old cultures grown on rye agar B at 18°C. Genomic DNA (gDNA) was extracted using the Plant DNA Kit (DP320, Tiangen, China) according to the manufacturer’s instructions, quantified using Nanodrop-1000 (Thermo Fisher), and stored at − 20°C for further use.

SSR multi-locus analysis

Genomic DNA from each P. infestans isolate was amplified using 12 SSR markers (PI02, PI04, PI4B, PI63, PI70, SSR2, SSR4, SSR6, SSR8, SSR11, D13 and G11). PI02, PI04, PI63, PI70, were designed by Lees et al. (23), SSR2, SSR4, SSR6, SSR8, SSR11, D13 by Euroblight P. infestans genotyping protocol(https://agro.au.dk/fileadmin/euroblight/Protocols/12plex_genotyping_Protocol_Apr_2020.pdf), and PI4B and G11 by Knapova and Gisi (24). All SSR markers were fluorescent-labeled as follows: FAM for PI02, PI04, PI4B and PI63; VIC for PI70, SSR2, SSR4, SSR6, SSR8 and SSR11; and NED for D13 and G11. PCR was performed using 25 µl reaction mixture containing 1 µl of 20–50 ng DNA as the template,12.5 µl of 2 × PCR mix (Qingke Biotech, Beijing, China), 1 µl (10 µM/µl) each of the forward and reverse primers, and 9.5 µl of ddH₂O. The reaction were performed using the GeneAmp® PCR System 9700 (Applied Biosystems, USA), following the methodology described by Lees et al. (22), except the annealing temperature for PI4B (60°C). The 3500 genetic analyzer (Applied Biosystems) was used for PCR fragment detection. All PCR products were run on a sequencer according to the manufacturer’s instructions based on the GeneScan™ 500 LIZ® Size Standard (Part No. 4322682; Applied Biosystems). The SSR alleles were scored using GeneMapper v4.0 (Applied Biosystems).

Mitochondrial DNA (mtDNA) haplotype

All isolates listed in Table S1 were used for mtDNA haplotype determination following the method described by Griffith and Shaw (25). The mtDNA haplotype patterns are also presented in Table S1.

Mating type test

A small piece of cultured P. infestans was grown on rye agar A (21) with known reference P. infestans isolates presenting the A1 (88069) and A2 (88133) mating types. In a petri dish, three pairs of blocks were placed at equal distances as a triangle, and the plates were kept in an incubator at 18°C in the dark for 12–14 days. Then, a small piece of tissue was removed from the intersected edge to confirm the presence of oospores. For mating type determination, the test isolate forming oospores with the A2 reference isolate was designated as the A1 mating type. Similarly, the test isolate forming oospores with the A1 reference isolate was designated as the A2 mating type. Next, the test isolate forming oospores with both A1 and A2 reference isolates was designated as the A1A2 mating type. Then, the test isolate forming oospores alone without pairing was designated as the self-fertile mating type. Finally, the test isolate that did not form oospores with either A1 or A2 reference isolate was designated as the A0 mating type. All mating type determinations were replicated twice for each isolate.

Metalaxyl resistance determination

Metalaxyl resistance of each P. infestans isolate was determined using a piece of mycelium plug (diameter, 5 mm) grown on rye agar A supplemented with 10 mg/l metalaxyl (part No. 32012, Sigma Inc., USA). Test isolate mycelia that exhibited < 10%, 10%-60%, and > 60% growth on metalaxyl-supplemented medium compared with that on metalaxyl-deficient medium were considered sensitive, moderately resistant, and resistant, respectively (26). Each treatment was delivered in triplicate, and each isolate was duplicated.

Virulence test on detached potato leaves

For virulence test, sporangia were washed from fresh rye A medium using distilled water after 14 days of incubation at 18°C in the dark. The resulting spore suspension was diluted to 1 × 10⁵ sporangia·mL^− 1 and stored at 4°C for 2h to induce zoospore liberation. Then, 10 µl of the sporangial suspension was inoculated on a the abaxial surface of an at least 1-cm-long leaf (cultivars C1-C11 and Bintjie, harboring R1-R11 genes and non-R genes, respectively), and the leaves were placed in a moist incubator at 18°C under a 12 h of light (Lx = 2,000) and 12 h of dark. Leaf lesions were evaluated every day for seven days and scored under a microscope for the presence of sporangia to determine susceptibility and resistance.

Data analysis

One-way ANOVA was used for the statistical analysis of phenotypic differences. The polymorphism information content (PIC) values were determined using MVSP v 2.0. Genetic diversity among all populations was calculated based on Nei’s gene diversity, Shannon’s diversity index, and gene flow (Nm). Principal component analysis (PCA) was performed using NTSYS v 2.1. Excepted heterozygosity and mean allele number were estimated using POPGEN v 3.2, and pairwise F_ST values were calculated as described by Nei (27) to measure genetic differentiation over 12 years. Cluster analysis was performed using unweighted pair group method with arithmetic mean (UPGMA) in Clustal X 2.0 and MEGA 6.0.

Mating type determination

A total of 357 isolates were collected from Kunming and Dali between 2010 and 2021. All A1, A2, A1A2, and self-fertile mating type isolates were found in 2012, 2013, 2015, 2018, 2019 (Table 1). In isolates collected before 2012, the A1 mating type was dominant (68 out of 87), but since 2012, the mating type of the isolates dramatically changed from A1 to A2, A1A2, and self-fertile, with fewer A1 isolates. The four mating types represented 2.52%, 49.3%, 10.9%, and 12.89% of all isolates, respectively, during 2013–2021.

Table 1

Mating type distribution of 357 isolates of *Phytophthora infestans* in Dali and Kunming from 2010–2021 ^*Number of isolates (Dali/Kunming)
Year Mating type	2010	2011	2012	2013	2014	2015	2016	2017	2018	2019	2020	2021	Total
A1	13/13^*	15/14	8/5	2/0	0/0	0/1	1/1	0/0	0/1	1/1	0/0	1/0	77
A2	1/0	0/1	6/7	8/11	12/14	10/11	12/9	7/7	9/7	9/12	7/9	13/9	191
A1A2	0/0	0/0	1/1	3/2	0/0	3/1	0/0	6/5	4/4	2/1	6/2	0/0	41
SF	0/0	0/0	0/2	2/2	3/1	2/2	2/5	2/3	2/3	3/1	2/4	1/6	48

mtDNA haplotypes, metalaxyl resistance and pathotype

Most of the collected isolates (355 of 357) were Ia mtDNA haplotypes, and only two isolates collected from Dali (DL0703 and DL0708) in 2011 were IIa mtDNA haplotypes; no other mtDNA haplotypes were detected in the present study (Table S1). Moreover, over 40% of the isolates from 2010 and 2014, over 50% of the isolates from 2016 to 2021 and under 20% of the isolates from 2013 were metalaxyl-resistant, In addition, 51% of isolates collected from Dali (n = 110) and Kunming (n = 72) and five isolates representing the 13_A2 Blue genotype from the two regions were highly metalaxyl-resistant (Fig. 2).

Virulence tests against the R1-R11 genes of cultivars C1-C11 (CIP collection) and Bintjie were performed. The broad-spectrum pathotype of P. infestans (against R1-R11) was dominant, which shared 77 pathotypes, and almost all isolates could overcome all R genes, except R5 in 2013 (Fig. 3). All 357 isolates could easily infect and produce sporangia on Bintjie. There were no significant differences between the two sampling regions (P = 0.322).

SSR marker data of 357 isolates

A total of 58 alleles were detected across 12 SSR loci, with 5–10 alleles per locus, and 101 genotypic cluster branches were identified at a similarity index of 0.95 (Fig. 9). The most and second most frequent genotypes were represented by 69 and 42 isolates, respectively. The PIC values of all SSR markers, as determined in the present dataset, ranged from 0.233 (PI70) to 0.755 (PI04), and 242 distinct fingerprint patterns were noted among the 357 isolates (Table 2), distributed over 12 years in the ascending order of genotypes collected from both regions (Table 3; Fig. 4). The diversity at each locus was calculated using the Na and Ne values, which represented various ranges from Dali to Kunming. Based on the h and I values, the populations sampled from the two areas showed minor genetically significant differences from 2010 to 2021 but highly significant differences during the transition years. Over the 12 years, the F_ST values were in the range of 0.030–0.264 (Table 4), and the populations sampled in 2010, 2011, and 2012 were significantly different from those sampled in other years. However, after 2012, populations sampled over the remaining years were not genetically distinct from one another (Table 5). The mean genetic distance between the two regions in each year continuously increased between 2010 and 2012, between 2016 and 2017, and between 2019 and 2021 but decreased from the beginning of 2013 to 2016 and between 2017 and 2018 (Fig. 5). PCA of all SSR genotypes revealed high variability among the tested isolates in both regions and across all years. Specifically, in both regions, isolates from 2010–2012 were slightly more distant from those from other years (Fig. 6, 7). PCA of 357 isolates showed two distinct but overlapping dimensions, representing the two regions (Fig. 8). UPGMA cluster analysis of the data of 12 SSR loci was used to further investigate the differences over 12 years. One isolate from Dali in 2010, two isolates from Dali in 2013, one isolate from Dali in 2016, two isolates each from Kunming and Dali in 2018, four isolates each from Kunming and Dali in 2019, and one isolate from Dali in 2020 were placed in the upper part of the tree and rooted in the tree at a genetic similarity coefficient exceeding 95%. The most dominant genotype in the cluster was detected in both regions, and the 12-year collection included 191 isolates. The genotype representing 3928A contained five isolates, collected from 2017, 2018, and 2019. Metalaxyl sensitivity, moderate resistance, and resistance were correlated with genotype clusters (Fig. 9-A). Furthermore, in phylogenetic analysis, isolates belonging to the A1 (Fig. 9-B), A2 (Fig. 9-C), self-fertile (Fig. 9-D), and A1A2 (Fig. 9-E) mating type as well as total mating types (Fig. 9-A) were genotypically close. In addition, the phylogenetic analysis divide 357 isolates into five mainly clades, we name as YN_1, YN_2, YN_3, YN_4 and YN_5. There were some differences in mating types among the sampling years and two regions, Especially genotype 23 was only detected in Kunming and genotype 47 only in 2021 (Table S1).

Table 2

Diversity of SSR locus
SSR locus	H^a	PIC
PI02	0.688	0.611
PI04	0.194	0.755
PI4B	0.442	0.655
PI70	0.336	0.233
PI63	0.712	0.292
SSR2	0.190	0.332
SSR4	0.558	0.641
SSR6	0.412	0.501
SSR8	0.364	0.604
SSR11	0.672	0.704
D13	0.528	0.406
G11	0.784	0.688
H^a value from 0 to 1 representing no diversity to maximum diversity

Table 3

Information of most 5 dominant genotypes ^a: Genotype are existing in this region.
Genotype	Fingerprints	Percentage	Years related	Dali	Kunming
G15	00110001001110001000001000010 11000010001000000001100001000	5.60%	2010, 2012, 2013, 2015, 2016	+^a	+
G14	00110001001110001000001000010 01000010001000000001100001000	3.92%	2010, 2012, 2013, 2014, 2015	+	+
G24	00110000101110001000001000010 01000010001000000001100001000	3.08%	2015, 2016	+	+
G16	00110000101110001000001000010 11000010001000000001100001000	2.80%	2012, 2013, 2014, 2015, 2016	+	+
G17	00110001001110001000001000010 11000100001000000001100001000	2.24%	2013, 2014, 2015, 2016	+	+

Table 5

Genetic differentiation among 12 years calculated by F_st values
	2010	2011	2012	2013	2014	2015	2016	2017	2018	2019	2020	2021
2010	0.000^a
2011	0.131	0.000
2012	0.137	0.158	0.000
2013	0.040^*	0.029^*	0.096	0.000
2014	0.009^**	0.036^*	0.002^**	0.063	0.000
2015	0.016^*	0.047^*	0.008^**	0.070	0.069	0.000
2016	0.016^*	0.044^*	0.110	0.075	0.075	0.066	0.000
2017	0.051	0.170	0.148	0.122	0.124	0.111	0.101	0.000
2018	0.143	0.161	0.038^*	0.109	0.109	0.097	0.085	0.108	0.000
2019	0.030^*	0.060	0.137	0.106	0.108	0.095	0.084	0.110	0.095	0.000
2020	0.037^*	0.051	0.033^*	0.104	0.106	0.098	0.088	0.117	0.095	0.103	0.000
2021	0.009^**	0.028^*	0.020^*	0.177	0.177	0.166	0.157	0.188	0.169	0.172	0.169	0.000
^a Number means difference in genetic structure between each two years, 1 means significant difference but 0 means no difference (Nei, 1978).
^* Significance of difference (p < 0.05).
^** Significance of difference (p < 0.01).

In the present study, high population diversity in terms of genotype and phenotype was observed over 12 years. Four mating types, two mtDNA haplotypes, and various virulent races exhibited significant differences between the two study regions and across the sampling years, indicating that P. infestans populations in Yunnan have underwent continuous genetic changes over the 12 study years. Before 2012, isolates adopting the A1 mating type and presenting over 50% sensitivity to metalaxyl were dominant in both Kunming and Dali; however, a notable shift occurred after 2013; as such, fewer A1 mating type isolates was detected, and the A2, A1A2, and self-fertile mating type isolates as well as metalaxyl-resistant isolates became predominant in the population. However, the mtDNA haplotypes remained limited in this study, indicating that host selection, sexual reproduction, or environmental changes may be the major drivers of this shift. In addition, human-induced gene flow through seed tuber transportation may be a minor reason leading to dramatic phenotypic and genotypic changes (28). Our results are consistent with some previous reports (29, 30). Nonetheless, little is known regarding the true drift mechanisms underlying these change in mating type under field conditions. In this context, environmental factors, such as cultural materials, may play important roles in mating type shifts. For instance, the use of V8 juice induced the transition of the A2 mating type to the self-fertile (31). In the present study, we used V8 juice agar to maintain the isolates, but we found no significant changes from the A2 mating type to the self-fertile (A2: self-fertile = 191: 46). Furthermore, we did not detect oospores either on PLB lesions or in potato planting soil under microscopic observations in the field during the 12 study years. Further investigation of other characteristics of P. infestans in these regions is warranted to analyze specific genetic or environmental factors that may contribute to shifts in mating type. Climatic factors may contribute to the genetic diversity of microorganisms (32, 33). In 2012, continuous drought from March to October, as reported by the Yunnan Meteorological Administration (Public data), likely exposed P. infestans to stress, may leading to dominant clonal lineages changes. South-western China, especially in Yunnan, potatoes always planting year-round in small montanic fields, make very different microclimates even in same geographical location, previous studies have proved the adaptations of new PLB clonal lineages may due to climate changes, such as EU_41_A2 & EU_43_A1 expanded in Nordic and western EU contries (34).

Pathogenicity analysis showed that the R5 gene was absent in 2013 in both two place, which may be attributed to host selection. The correlation between hosts and genotype numbers also support this hypothesis (Fig. 10). From 2010 to 2012, the main potato cultivars included Cooperation 88 (resistant to PLB), Dianshu 6 (moderately resistant to PLB), and Lishu 7 (resistant to PLB) in Kunming. However, from 2013 to 2015, the main cultivars became Xuanshu 2 (sensitive to PLB) and Kunshu 2 (moderately resistant to Metalaxyl); thus, pathogens faced decent host pressure in these years compared with that in the previous years, leading to the nonexistence of R5 in 2013. Surprisingly, Cooperation 88 and Lishu 7 remained the main cultivars planting in Dali for 12 years, and the lack of R5 in 2013 in this region, may be major attributed to fungicide abuse, resulted in a decrease in the number of methomyl resistant strains compared to the year of 2012 and 2014.

Furthermore, using 12 SSR markers screened in this study, a total of 58 alleles were detected among the tested P. infestans isolates. Most of the markers were useful in the differentiation of genotypes among the test isolates. The smallest increase in the genetic diversity of P. infestans was noted at the beginning of 2013 in both study regions, strongly indicating that the clonal lineages adopted asexual propagation in their previous life cycle; this notion is supported by previous reports (2). The cluster root represented 11 isolates in 2010, 2013, 2016, 2018, 2019 and 2020, with various mating types, consistent metalaxyl resistance and mtDNA haplotype, indicating the possibility of host adaption and limited sexual reproduction in 2010, which may be responsible for the subsequent genetic drift. Moreover, phylogenetic analysis revealed a certain correlation between genotype and mating type; some A1 mating type isolates were placed on three major branches (Fig. 9-B), A2 mating type isolates on four branches (Fig. 9-C), self-fertile mating type isolates on two branches (Fig. 9-D), and A1A2 mating type isolates on three branches (Fig. 9-E), indicating that the genotypes were restricted relative to the collection years. Metalaxyl sensitivity was also linked to some genotypes detected in the present study. Previous studies have shown that metalaxyl resistance is conferred by a single locus (35), and the 12 SSR markers used in the present study may be linked to this functional locus in our pathogen populations; however, these results are not consistent with previous reports (7, 8, 36, 37). To further understand the correlation between genotype and phenotype, additional SSR markers and a wide range of isolates must be tested.

Migration may be minor reason for genetic change. The highly aggressive genotype EU_13_A2 Blue, which presents the mtDNA haplotype Ib, has been spreading in Europe since 2012. This genotype was first detected in 2004, and during 2006–2007, it became dominant in the Netherlands, France, UK, Germany, Belgium, eastern Africa, Turkey and Pakistan (8, 38–40). According to a previous study, this genotype has also been detected in the Yunnan and Sichuan provinces of China (7). Potatoes transportation across various localities may lead to the migration of highly aggressive phenotypes and genotypes along with the host, thus aggravating the problems of late blight and leading to population change. Here, the detection of five isolates collected from the two regions over the 12 study years indicates the migration of pathogens through the transportation of potato tubers between these regions or of seed tubers from neighboring provinces or southeastern Asian countries for several years, creating a vicious circle of pathogen adaption to local potato or tomato varieties. In the present study, crossing groups based on collection years in PCA analysis also suggested that P. infestans isolates in Yunnan have been frequently transported from or to the two study regions, as well as other neighboring southeast Asian countries (e.g. Vietnam and Myanmar) or southwestern Chinese provinces via contaminated tuber seeds, but may not from northern provinces. Furthmore, oddly, among the 357 isolates, only two isolates presented the mtDNA haplotype IIa, while the rest presented the mtDNA haplotype Ia, indicating that the isolates did not complete migrate from northern China, Southeast Asia or Europe but rather prefer asexual life cycle, given the significant genetic differences between isolates from Yunnan and northern provinces (28).

In this study, five isolates presenting the 13_A2 Blue genotype with the mtDNA haplotype Ia were detected in 2017; however, they did not become dominant populations in the two regions after the year of 2018, indicating that the mating type shift did not occur due to the asexual life cycle of the 13_A2 Blue clonal lineage but rather due to isolated evolution in neighboring regions. Surprisingly, after 2019, the same 13_A2 Blue genotype was no longer detected, the same situation as Europe from 2021(41). In addition, based Asiablight database, EU_13_A2, EU_8_A1, US_11_A1, US_1_A1 and SIB_1_A1 were as dominated lineages around southern and northern China during 2008–2019, these genotypes were not detected in two sample regions except EU_13_A2, also reflected migration is not a major reason, further studies are warranted to confirm the existence of our 5 genotypes using additional DNA markers.

Overall, the present study indicates that P. infestans isolates from Kunming and Dali in Yunnan mainly reproduce asexually, although limited sexual reproduction or gene flow may occur, enhancing population genetic diversity. Such changes allow P. infestans to rapidly overcome host resistance and adapt well to the local environment. Meanwhile, the novel virulence gene flow over the generations may break down local host resistance. To effectively control PLB in Yunnan, climate change and pathogen population structure must be continuously monitored and the adaptive potential of pathogens must be promptly assessed, which would allow for the optimization of fungicides and prediction of PLB epidemic trends. Notwithstanding, although it is difficult to truly understand the reason for mating type drift in P. infestans in Yunnan, increased genetic diversity during 2010–2021 may result from gene flow, particularly agriculture-mediated gene flow, which can be regulated through the use of healthy potato seeds and implementation of strict quarantine protocols, in addition to reasonable use of fungicide and selection of appropriate cultivars in different regions.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (grant numbers 31660503, 32060499 and 32101665) and the Yunnan Fundamental Research Projects (grant number 202301AS070010, 2019FB023 and 2202201AT070026). The authors also thank Professor Z. D. Tian (Hua Zhong Agriculture University, China) for providing the reference isolate 3928A and Professor L. Y. Guo (China Agriculture University, China) for providing the reference isolates 80029 and 88133. The authors are grateful to Miss Y. Q. Miao (Yunnan Normal University, China) for isolate collection during 2010-2014.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Competing interests

The authors declare that they have no competing interests.

Funding

National Natural Science Foundation of China (grant numbers 31660503, 32060499 and 32101665); Yunnan Fundamental Research Projects (grant number 202301AS070010, 2019FB023 and 2202201AT070026)

Authors' contributions

W T, JML conceived and designed this research, JSL, CT, MLW and DHH collected samples and analyzed the data. ZL and WJZ prepared the figures and tables; WT and JML wrote the manuscript; WT, JML and JSL revised the paper. JL, HYW and CHL are the project leaders and corresponding authors. All authors read and approved the final manuscript and agreed to submit.

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Table 4 is available in the Supplementary Files section.

No competing interests reported.

TableS1.docx
Table S1 Information of isolates collected from Dali (DL) and Kunming (KM) during 2010–2021.
Table4.docx

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Mating type revulsion and dynamic genetic diversity of Phytophthora infestans during 2010–2021 in yunnan, China

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Abstract

Figures

INTRODUCTION

MATERIAL AND METHODS

Strain collection, isolation, and culture conditions

DNA extraction

SSR multi-locus analysis

Mitochondrial DNA (mtDNA) haplotype

Mating type test

Metalaxyl resistance determination

Virulence test on detached potato leaves

Data analysis

RESULTS

Mating type determination

mtDNA haplotypes, metalaxyl resistance and pathotype

SSR marker data of 357 isolates

DISCUSSION

Declarations

References

Tables

Additional Declarations

Supplementary Files

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