1. Introduction
Late blight, caused by a hemibiotrophic oomycete pathogen
Phytophthora infestans (Mont.) de Bary (1876), is one of the most devastating diseases affecting potato (
Solanum tuberosum L.) and tomato (
Solanum lycopersicum Mill.) worldwide [
1,
2]. The disease has a significant socio-economic impact on potato and tomato production, causing an estimated annual loss of USD 6.7 billion to potato and 100% crop loss to tomato in some parts of the world [
3]. The first outbreak of late blight was recorded 170 years ago in the USA, and it subsequently spread across continents to Europe and other parts of the world [
4].
P. infestans is a persistent threat to potato and tomato cultivation worldwide due to its aggressiveness, host adaptability, high mutation rate, and transportation of infected vegetative material [
5]. The origin of
P. infestans is disputed, but it is believed to have originated in either South America [
6] or central Mexico [
7]. The genotypic and phenotypic characterization of populations of
P. infestans has defined the role of certain clonal lineages in disseminating the pathogen across potato-growing regions [
8,
9]. The infamous Great Irish Famine of the 19th century was caused by the FAM-1 lineage [
10], which was subsequently displaced by the US-1 lineage during 1946–1955. Both of these lineages, FAM-1 and US-1, were later displaced by more aggressive and distinct clonal lineages that migrated from Mexico to other parts of the world [
10]. Such rapid shifts in the population structure of
P. infestans have been continued with genetically diverse populations.
The algal-like oomycete
P. infestans is a diploid heterothallic pathogen that requires association between mating types A1 and A2 to produce long-lived oospores [
7]. Sexual progeny from the germinated oospores results in the formation of genetically diverse populations. The dominating
P. infestans populations in Western Europe comprise more aggressive clonal lineages such as 13_A2 and 6_A1 compared to more diverse sexual populations in central Mexico and Nordic countries [
11]. Among A2 mating types, 13_A2 is a highly aggressive and the most dominant metalaxyl resistant lineage that was first reported in 2004 in the Netherlands and Germany [
12]. Later, during 2006–2009, it appeared in Poland [
13] and the United Kingdom [
12]. Subsequently, it caused severe outbreaks in potato and tomato crops in China [
9] and India by replacing prior populations of US-1 and related genotypes [
3]. Similarly, during a 2008–2014 survey, the 13_A2 genotype accounted for the majority of the population in Algeria, followed by the 2_A1 and 23_A1 genotypes [
14]. In Africa, the first
P. infestans epidemic was reported in 1941 in Kenya, and a year later, it was reported in Uganda. It spread across borders in the successive years, into the Congo in the west and Tanzania in the south [
15].
P. infestans strain 2_A1 (of European origin) was recently found to be dominating in east African agro-ecological regions, including Burundi, Kenya, Rwanda, Tanzania, and Uganda. The introduction and prevalence of the A2 mating type of
P. infestans in North Africa may be due to high imports of seed potato from European countries and resulted in the introduction of new European strains in Algeria, Egypt, Morocco and Tunisia [
14,
16], whereas in North Africa, only the A1 mating type has been persistently found [
17]. Egypt is one of 15 countries that exported 5.2% of unprocessed potatoes in 2020, worth USD 221.9 million [
18].
P. infestans was presumed to have been present in Egypt since at least 1941 [
19], and A1 and A2 mating types, as well as self-fertile isolates, have previously been identified in Egypt [
20,
21,
22]. Nevertheless, the distribution of various mating types varied from year to year in Egypt [
20,
21,
22,
23,
24,
25].
Understanding the population structure, evolution, recombining abilities and epidemiology of
P. infestans in a particular region over time helps decision making in integrated pest management (IPM). In addition to its epidemiological impact, sexual recombination is another pivotal factor that contributes to the rate of pathogen adaptation [
26]. Monitoring the A1 and A2 mating-type ratios is important to know the potential extent of sexual recombination and thus the risk of long-lived oospores serving as primary inoculum sources [
8]. Microsatellite markers are extremely useful for deciphering the population structure, characterizing the multilocus genotypes, genetic mapping, and estimating the evolutionary processes [
27]. An efficient one-step multiplex simple sequence repeats (SSR) protocol employing twelve SSR markers for high-throughput screening of
P. infestans populations throughout the world has been developed [
9]. Effectors are proteins that can be seen as the pathogen’s key weapon to defeat the host’s defense mechanisms [
28]. Effector genes condition the susceptibility or resistance of hosts with known R-genes and can both facilitate the infection (virulence factors or toxins) of a host or trigger defense responses (avirulence factors). Thus, phenotypic and genotypic characterization of the pathogen strains may help in the development of more specific disease management strategies. The objectives of this study were to (1) understand the distribution of different mating types of
P. infestans from potato and tomato in Egypt, (ii) characterize the Egyptian population of
P. infestans using SSR microsatellite markers, (iii) determine metalaxyl sensitivity and virulence profile and (iv) to study the presence and diversity of key effector genes.
4. Discussion
This study of the population of P. infestans in Egypt over three consecutive cropping seasons has identified two dominant clonal lineages of European origin (13_A2 and 23_A1). Their phenotypic (virulence and mating type) and genotypic (SSR and effector diversity) traits have been examined and are discussed in relation to other studies.
Isolates belonging to both the A1 and A2 genotypes were detected in all six governorates of Egypt. However, self-fertile isolates were detected in five governorates: Menofia, Beheira, Gharbia, Kafr El-Sheikh and Dakhilia. The proportion of the A1 mating type in this study ranged from 38–70%, indicating a dynamic population; however, no clear trend was apparent over the three seasons examined. The establishment of A1 mating type in the Egyptian population corroborated previous findings that the A1 mating type has not only established itself in the Egyptian environment [
45], but it also exhibits high divergence and polymorphism [
46]. Another study is in line with our findings found that 15% of 162 isolates of P. infestans sampled during 2005–2006 from Beheira governorate and the surrounding area were A2, 84% were A1, and only two isolates were self-fertile {El-Korany, 2008 #12620]. Samples collected in 2015 to 2016 identified 67 A1, 12 A2 and one self-fertile mating type from Egypt [
46]. We examined the infected plant tissues for the presence of oospores, but none were seen in samples from which self-fertile isolates were recovered. However, a low number of oospores were noted in a single blighted stem of cv. Diamont in Tamalay, Menofia Governorate in January 2011 at the end of the growing winter season. Oospores of
P. infestans have been reported in field-grown potato and tomato in several countries [
47,
48,
49,
50]. This indicated that there might be other factors affecting oospore formation in situations where only one mating type is reported under natural infection in fields in Egypt. Investigating such factors may open up a plethora of resources for combating the disease and could be the subject of future study.
P. infestans is heterothallic and requires two mating types, A1 and A2, for sexual reproduction [
51]. However, the term heterothallic is not absolute when applied to this pathogen. There have been many reports of oospores in single isolate cultures of
P. infestans [
4,
52,
53,
54].
The current survey identified a dynamic population amongst isolates of
P. infestans sampled from Egypt. The cluster analysis (
Figure 4B) grouped all the isolates into two main lineages. The first one contained twenty-nine 13_A2 blue clonal lineage isolates. The 13_A2 lineage was first sampled in the Netherlands in 2004, and isolates with this genotype are highly aggressive, spread rapidly and now dominate
P. infestans populations in large parts of Europe [
12]. Some studies show that isolates of the 13_A2 genotype had a shorter latent period, were more aggressive and are able to overcome resistance to some potato cultivars [
55]. It was therefore considered a new threat to potato production, and its spread was intensively monitored in Europe (
www.euroblight.net, accessed on 2 February 2022). The lineage blue 13_A2 has spread to other potato- and tomato-growing areas of the world and has caused severe outbreaks of late blight on tomato in south-west India [
3]; blue 13 lineages have been predominantly found in southwestern China [
9,
56]. Interestingly, the current study is the earliest date that 13_A2 has been reported in Egypt.
In this study, three distinct sub-clonal lineages of 13_ A2 were detected. This is comparable with the levels of diversity recently reported in Turkey [
57], but lower that the twenty sub-clonal types reported in Cyprus [
58]. The forms 13_A2-5 and 13_A2-43, previously reported from Europe, have been present in Egypt at least since 2009 [
12,
56]. The third sub-clonal lineage was 13_A2-84, which differed at three SSR loci and contained a new allele combination (266-272 bp) at the Pi02 locus. This sub-clonal lineage may have been generated under Egyptian selection pressure conditions and may be considered high-temperature-tolerant, as numerous outbreaks were observed in May when the temperature ranged from 25–35 °C during day and 18–22 °C during night. Nonetheless, it is far from conclusive and needs further elaboration. Tolerance to high temperatures was reported on A1 and A2 lineages in Tunisia [
59] with a suggestion that A2 isolates were more tolerant. It was not clear whether the A2 isolates in the Tunisian study were of 13_A2 but they were aggressive and metalaxyl-resistant [
59]. All the 71 13_A2 blue lineage isolates tested in this study were fully resistant to metalaxyl, which is consistent with other reports [
12,
60] and has implications for use of this fungicide in Egypt.
The second cluster in the dendrogram contained 55 isolates of 23_A1, and within this there were ten subclones defined. These data are consistent with other studies that have reported its presence and sub-clonal diversity in southern Europe, Tunisia and Algeria [
14,
61,
62]. In agreement with other work [
61], genotype 23_A1 is identical to US-23, and both are reported to be tomato-adapted. Its spread in Europe may have mirrored its spread in the US via trade in tomato plants [
61]. It is unclear how this lineage was imported into Egypt, but it may be related to tomato production. It is speculated that the 23-A1 clonal lineage adapted to the Egyptian environmental conditions due to its widespread distribution in both potato and tomato crops.
Lastly, a single isolate (EG-6) that was unrelated to 13_A2 and 23_A1 was detected. It had three novel alleles that were not found in the other two lineages (D13 (118 bp), G11 (204 bp), and SSR6 (242 bp). This genotype has not been reported in other studies or in the large EuroBlight database. The origin of such novel genotypes is unclear. It may be a local recombinant formed from an oospore germination event in an Egyptian crop or represent a genotype imported with potato seed from another part of Europe.
The genome of
P. infestans consists of an extensive expansion of specific families of secreted disease pathogenicity effector proteins (>700), which are coded in the mobile element of the genome [
63]. The pathogen effector, which is a product of an avirulence (Avr) gene, interacts with the corresponding R protein in the plant. The effector proteins can target different sites in infected host plant tissue [
64]. These protein–effector interactions are studied to better understand the underlying molecular mechanisms of late blight resistance. All oomycete avirulence genes discovered to date have an RxLR-motif (RxLR=arginine, any amino-acid, leucine, arginine) [
44]. The
P. infestans effectors Avr2, Avr3a, Avr4 and Avr-blb1 are the most studied effectors and belong to the RxLR group [
43,
44,
65].
To understand the underlying mechanisms of how
P. infestans evades recognition and overcomes resistance genes in potato, the presence/absence of five effector genes that are responsible for virulence or avirulence activity on potato clones carrying R1-R4 genes was screened. In almost every case, the evidence from the effector characterization (
Figure 6) matched the results of the differential screening (
Figure 3). Blue lineage 13_A2 isolates were virulent on the potato differential carrying R1, and their virulence was associated with the deletion of the Avr1 gene, as reported previously [
12]. In contrast, the Avr1 locus was present and amplified in the 23_A1 and misc. isolates. Unexpectedly, despite the presence of avr1, the 23_A1 isolates were also virulent on R1. The avr1 sequences recovered in this study from 23_A1 and the misc. isolate matched other isolates reported to be avirulent [
66], so the cause of the virulence is unclear. Perhaps, like with avr2 [
42], the gene is not expressed and therefore not recognized by R1. Consistent with studies on other clonal lineages [
42], Avr2 was amplified in all six 23_A1 and one misc. isolate tested. No SNPs were detected amongst these isolates resulting in the avirulent form, which was recognized by the R2 plant. Conversely, Avr2 was absent in all eight 13_A2 lineage isolates tested and the 13_A2 isolate was virulent against R2. Unexpectedly, in one 13_A2 isolate (EG-96), a product was amplified. However, it was shown to match the avr2-like gene, indicating a non-specific amplification, which is consistent with other studies and likely a PCR artefact due to primer mismatching [
42]. The avr2-like gene was amplified from isolates of all tested samples, and two SNPs were identified that matched other work [
42] did not influence the virulence test results. Consistent with other studies [
43] Avr3a was present in all tested isolates and contained the homozygous EM or heterozygous EM/KI virulent forms, which corresponded to the virulence test results against R3a in this study. Avr4 was present in all nine tested isolates. Both homozygous and heterozygous forms of Avr4 were virulent, with SNPs matching those previously reported [
44]. The findings in the current study widen our understanding of the effector diversity in the 23_A1 lineage. Such data are useful in the long-term understanding of host–pathogen interactions and are critical to the future deployment of durable forms of late blight resistance [
65].