Morphological and Molecular Variability of Alternaria solani and Phytophthora infestans Causing Tomato Blights

Alternaria solani and Phytophthora infestans cause early and late blight diseases in tomato and potato, respectively. A. solani can survive for more than a decade in the soil, seed, or in plant residues at optimum temperature. The pathogen exhibits high molecular and genetic variation between isolates from potato and tomato plants, in different countries. Morphological studies reveal separate conidia borne singly on simple conidiophores. Spores are elongated, muriform, beaked, septate, and dark coloured. The mycelia are branched and septate. A. solani demonstrated a high genetic variability among isolates originating from the United States, Greece, Cuba, Canada, Russia, Turkey, South Africa, Brazil, and China based on vegetative compatibility groups and molecular markers (random amplified polymorphic DNA markers, random amplified microsatellite markers, and amplified fragment length polymorphisms). Different morphological and molecular variations indicate the presence of variability among the isolates. On the other hand, P. infestans is a diploid, obligate, heterothallic, and biotrophic oomycete, whose asexual lifecycle is characterized by alternating phases of sporangia germination, hyphal growth, and sporulation. The mycelia of P. infestans is coenocytic, multinucleate, and aseptate although the cross walls do not form in old cultures. Sporangia are borne singly on the branch tips of the alternately branched sporangiophore. Sporangium is hyaline and lemon shaped with a papilla at the distal end. Mating types A1 and A2 with different clonal lineages have been discovered in various parts of the world indicating variation in the species.


Introduction
Alternaria solani causes early blight disease in tomato. Te pathogen belongs to the phylum Ascomycota, class Deutromycetes, and order Moniliales [1]. A. solani is a soil inhabiting fungus and spreads to host plants through air and rain splash [2]. Te pathogen is among the large-spored group of the genus Alternaria which is distinguished by separate conidia borne singly on simple conidiophores. Its spores are elongated, muriform, beaked, septate, and dark coloured, while the mycelia are branched and septate [3]. Te fungus survives as parasitic and as a saprophytic organism [4]. Early blight disease was frst identifed on potato, and the causative pathogen was named Macrosporium solani and was later classifed under the genus Alternaria based on the spore development in chains in a culture medium [5]. Te pathogen can survive for more than ten years in the soil, seed, or in plant debris at optimum temperature [6]. Te fungus produces toxins such as alternaric acid, altersolanol, macrosporin, and zinniol that act on the protoplasm of the host and distract plant defense mechanisms [2].
A. solani exhibits a high genetic variation between isolates from potato and tomato plants and from diferent countries due to pathogen specialization and pesticide resistance [7]. A. solani fungal isolates from diferent host plant species vary in aggressiveness, physiology, and genetic diversity when inoculated in diferent plants [7]. When environmental conditions are favourable, the conidia germinate and form one or more germ tubes which penetrate the epidermal cell of the host through the stomata or directly by means of appressoria or wounds by hyphal growth [2]. Te pathogen colonizes the host by degrading the cell walls of the host by producing toxins that destroy the cells of the host, therefore enabling them to acquire nutrients from the plant cells [8]. Te toxins such as alternaric acid, altersolanol, macrosporin, and zinniol act on the protoplasm of the host and disrupt physiological processes that sustain the health of the plants [2]. Te pathogen can survive for more than a decade in the soil, seed, or in plant residues at optimum temperature [9]. Te pathogen causes early blight disease on tomato plant foliage. Te disease infects all above ground parts of the tomato plant at all stages of the plant growth and development [10]. Early blight disease can cause 79% crop loss in tomatoes [11]. Quantity losses to early blight disease on tomato amount to 2.15% in resistant varieties and 42.75% in susceptible varieties [12].
Phytophthora infestans causes late blight disease in tomatoes and other solanaceous plants [13]. Te genus name Phytophthora is coined from two Greek words; "Phyto" meaning plant and "phthora" meaning destroyer [14]. Te pathogen results in massive destruction on tomatoes [15]. P. infestans belongs to the kingdom: Chromista, phylum: Heterokontophyta, class: Oomycota, order: Peronosporales, and family: Pythiaceae [16]. P. infestans is a diploid, obligate, heterothallic, and biotrophic oomycete, whose asexual lifecycle is characterized by alternating phases of sporangia germination, hyphal growth, and sporulation [17]. Morphologically, the pathogen is similar to fungi but phylogenetically related to brown algae or diatoms because their sexual oogonia and antheridia resemble those of brown algae. Te pathogen is sometimes referred to as water mould because of its special adaptations to water habitat [18]. Te pathogen's cell wall is made of cellulose and other glucans unlike fungi whose cell walls are made of chitin [19]. Te genus Phytophthora has no ability to synthesize thiamine and sterol because it lacks the synthesis pathways and therefore acquires these essential compounds from the host plant [17].
P. infestans is a diploid, obligate, biotrophic, and heterothallic pathogen with two mating types: A1 and A2 [20]. Te pathogen is an oomycete, and its asexual lifecycle is characterized by alternating phases of hyphal growth, sporangia germination, and sporulation [21]. Sexual reproduction results to oospores that are thick walled to enable them overcome harsh environmental conditions such as cold, chemical fumigations, and microbial degradation, thus conserving the inocula for the subsequent years [22]. Te pathogen has low levels of diversity, and its population structure and distribution are infuenced by host preference [23]. Te mycelia of Phytophthora is coenocytic, multinucleate, and aseptate although the cross walls do not form in old cultures [24]. Sporangia are borne singly on the branch tips of the alternately branched sporangiophore [25], while the sporangium is hyaline and lemon shaped with a papilla at the distal end [26].
Sporangia are released from the sporangiophores by twisting and popping of the sporangiophore, and they are disseminated by air currents [27]. Te zoospore formation within the sporangia is temperature dependent. Vegetative spores and mycelia can exist and remain infectious in plant debris for not less than one week and continue to cause infections when they are in contact with susceptible plant tissues [25]. Te pathogen can grow on selective culture media such as rye agar, lima bean agar, pea agar, corn seed agar, and V-8 agar [19]. In the culture, P. infestans grows slowly and the mycelia are white and fufy although some isolates have lumpy appearance [17].
Te sexual cycle of P. infestans occurs when mating types A1 and A2 meet and release hormones that trigger the formation of oospores [15]. For the production of oospores, both the A1 and A2 mating types are needed. However, the presence of the two types is not a guarantee of sexual reproduction since some levels of incompatibility exists [28]. Te mating types are compatibility types, diferentiated by mating hormones, but are not dimorphic forms. When the mycelia of type A1 and A2 mating types interact, the mating hormones induce gametangial formation in the opposing mating types, initiating sexual propagation by means of oospore formation [29]. Te mycelia undergo meiosis during gametangia formation to form antheridia and oogonia that are haploid. Te antheridium fuses with an oogonium during the sexual life cycle to form a diploid oospore [30]. Sexual reproduction results to gene recombination that generates new virulent strains, thus complicating the management of the disease [31].

Morphological Diversity of Alternaria solani
2.1. Colony Radial Growth. Studies have reported that the colony growth varies between A. solani races subjected to the same growth conditions. According to Kumar et al. [32], the radial growth ranged from 14.9 mm to 57.7 mm among eleven isolates investigated. Few isolates grew fast within the frst four days, others increased growth in days 6-7, while another lot increased its growth on the last days of the experimental period [32]. Nikam et al. [33] investigated the radial growth of eight A. solani isolates and reported radial growth ranging between 65.80 mm and 88.50 mm. Similarly, Nafsa and Javed [34] evaluated seven isolates of A. solani and reported their growth to range between 50 mm and 88 mm within a period of seven days. One of the isolates had the slowest growth and attained a radial growth of 50 mm by the end of the experimental period. One of the isolates grew to 60 mm, two with 65 mm, one with 79 mm, and two with 88 mm. Diferences in radial growth among isolates signify variability. Te radial growth obtained from two isolates studied by Rahmatzai et al. [35] was 79 mm for isolate one and 61 mm for isolate two.

Colony Growth Pattern.
Te fungal colony growth pattern also varies between isolates which is an indication that there are many strains. For example, according to Kumar et al. [32], some of the isolates had circular or irregular growth margins, while some had rough and others with smooth margins. Among the 19 fungal isolates studied by Mugao et al. [36], majority of them (12) had a growth margin that was irregular, while the others (7) had a circular growth margin. Among eight isolates studied by Nikam et al. [33], the growth margins were either circular or regular and either smooth or rough. Some had zonation, while others were without zonation. Results of Nafsa and Javed [34] also showed that the growth margins of the seven isolates studied were regular and smooth and there was none with an irregular growth margin. Loganathan et al. [37] studied growth patterns of 5 A. solani isolated and reported that 3 had a regular growth pattern, while 2 had an irregular growth pattern.

Colony
Colour/Pigmentation. Te colour of A. solani varies among races. According to Kumar et al. [32], some of the A. solani isolates appeared black, others had yellow pigmentation and appeared greenish, while others were brownish black. Similar results were reported by Mugao et al. [36], and after evaluating nineteen isolates, the colony colour varied from being greenish brown, dark brown, grey, to being greyish brown. Nikam et al. [33] evaluated the morphology of eight A. solani isolates, and only two out of the eight isolates had a similar colony colour which was brownish black. Te other six isolates had diferent colony colours (black, dark grayish, olivaceous black, pinkish red, light grey, and creamish white). According to Nafsa and Javed [34], the seven isolates studied had diferent colony colours. Two of them were blackish brown, three were dark brown, and two were black in colour. Studies of Loganathan et al. [37] also revealed variations in A. solani colony colour. Out of the 5 isolates studied, four of them had grey colony colour, while one was yellowish grey. Upadhyay et al. [38] revealed a high degree of variation of colony colour of 32 isolates studied. Among them, two were black, 3 were grey, 2 were grey with white pigmentation, and 3 were grey with cream pigmentation among many other colour combinations. Results of Rahmatzai et al. [35] revealed reddish black pigmentation and brownish black pigmentation on the two isolates studied, while black with pale red pigmentation and dark grey to black with dark pigments were shown on two isolates studied by El-Ganainy et al. [39].

Conidial Length and Tickness.
Research has shown that conidial length and thickness of A. solani varied between diferent isolates, indicating the presence of diferent races. Reports of Kumar et al. [32] showed that the length of the conidia also varied ranging from 150 to 300 μm, while thickness ranged between 15 and 19 μm. Ahmad [40] studied the conidial morphology of A. solani and discovered that some have large conidia of 175 μm. Studies conducted by Nafsa and Javed [34] revealed conidial width ranging between 10 and45 μm. According to Loganathan et al. [37], the conidial length of the fve isolates studied ranged between 80 and 97 μm and the thickness ranged between 23 and 32 μm. Two of the A. solani isolates studied by Rahmatzai et al. [35] showed varied conidial length and width. Te frst isolate had a higher conidial length of 25-44 μm and a width of 7-15 μm, while the second one had 20-30 μm length and 5-13 μm width. Te isolates studied by Naik et al. [41] showed 19.5-70 μm conidial length and 6-22 μm width. Other isolates had conidial length ranging from 70.63 to 77.61 μm and a width of 16.94-25.05 μm [39].

Conidial Colour.
Research has shown that the colour of the fungal conidia varied with isolates indicating variability. For example, according to Kumar et al. [32], the conidia colour varied from dark muriform, pale golden, to olivaceous brown. Tese results were in consonance with those reported by Nafsa and Javed [34]. However, Rotem [8] reported that the conidia of A. solani are brown, grey, or dark coloured with high content of melanin that protects them against diverse environmental conditions including resistance to microbes and hydrolytic enzymes.

Conidial Beak Size and Shape. Studies on A. solani
have revealed that their beak sizes and shapes vary within races. According to Ahmad [40], beak length ranged between 47 and 65 μm. Some have their beaks branched, while others have unbranched beaks [32]. Mugao et al. [36] evaluated the beaks of 19 isolates where twelve isolates had elongated and unbranched beaks. Six of the isolates had slender, short, unbranched beaks, while one had an elongated branched beak. Te report of Nikam et al. [33] also showed that the beak sizes also varied with some having elongated beaks, while others had short beaks. Seven strains of A. solani studied by Nafsa and Javed [34] revealed a beak size ranging between 10 and 50 μm. According to Loganathan et al. [37], the beak length of the 5 isolates studied ranged between 60 and 89.3 μm. Rotem [8] reported that the length of the beak size of A. solani conidia ranged between 139 and144 μm and the beak width ranged between 12-20 μm. According to Simmsons [42], the conidial sizes were 120-296 μm length and 12 and 20 μm width. Rahmatzai et al. [35] reported that the two isolates studied had a varying beak size of 6-10 μm and 4-8 μm, respectively, while El-Ganainy et al. [39] had isolates with beak length ranging between 27.91 and 116.61 μm.

Conidial Septation.
A. solani isolates evaluated by diferent researchers revealed that the conidia were septate, but the number of septa varied between isolates. For example, according to Kumar et al. [32], the number of conidia septa ranged between 9 and 11 transverse septa and 1-4 longitudinal septa. Mugao et al. [36] studied the conidia septa of 19 A. solani isolates and concluded that the number of conidia septa also varied between the isolates with thirteen of the isolates having four transverse septa but no longitudinal septa. Te study further revealed that three of the isolates had three transverse septa and out of the three, one of the isolates had one-longitudinal septa, but the other two did not have. Nikam et al. [33] reported that the conidial septation varied between the isolates with the transverse septa ranging between 5 and 12, while the longitudinal septa ranged between 1 and 4. Seven isolates of A. solani studied by Nafsa and Javed [34] also showed septa variation. Te longitudinal septa ranged between 3 and 10, while the transverse septa ranged between 1 and 3. However, results of Simmsons [42] and Singh [4] showed that the conidia contained 5-10 transverse septa and 1-5 longitudinal septa. Rahmatzai et al. [35] reported varying conidial septation from the two isolates studied. Te frst isolate had 2-11 International Journal of Microbiology 3 transverse septa and 1-3 longitudinal septa, while isolate two had 2-8 transverse septa and 1-3 longitudinal septa. According to Naik et al. [41], the transverse septa of A. solani studied ranged between 2 and 7, while the longitudinal septa ranged between 1 and 4. However, two isolates studied by El-Ganainy et al. [39] had more longitudinal septa (5-11 and 2-5) than the transverse septa (3-7 and 0-3).

Molecular Diversity of Alternaria solani
3.1. PCR Amplifcations. Alternaria solani pathogen exhibits a high genetic variation between isolates from tomato and potato plants in diferent countries [32]. A. solani demonstrated a high genetic variability among isolates originating from the United States, Greece, Cuba, Canada, Russia, Turkey, South Africa, Brazil, and China based on vegetative compatibility groups [43] and molecular markers (randomly amplifed polymorphic DNA markers, randomly amplifed microsatellite markers, and amplifed fragment length polymorphisms) as reported by Pérez Martínez et al. [44]. Nikam et al. [33] conducted a molecular study of 8 isolates using RAPD-PCR analysis, and the amplifcation process regrouped them into two groups, and each group had an 80% similarity coefcient. PCR products obtained by Nafsa and Javed [34] on seven strains revealed variations in band sizes ranging between 518 and 549 bp using ITS primers. Previously, A. solani species yielded band sizes ranging between 400 and 600 pb using ITS primers [45,46]. Reports of Loganathan et al. [37] showed that the ITS regions of fve isolated amplifed produced a similar band size of 580 bp. According to Upadhyay et al. [38], 32 isolates of A. solani DNA amplifed using diferent primers yielded band sizes ranging between 200 and 1500 bp, an indication of variability among isolates.

Gene Sequencing to Study Genetic Diversity of A. solani.
Diferent countries have used diferent methods to study genetic diversity of A. solani. For example, Naik et al. [41] studied genetic diversity among four A. solani isolates and reported that three of the isolates had 73.78% similarity, while the other one shared 45% genetic similarity, indicating distinct polymorphism. In Europe, A. solani carries two types of mitochondrial DNA referred to as genotype 1 (GI) and genotype 2 (GII) [47]. Seven isolates studied by Nafsa and Javed [34] were reported to have diferent accession numbers (MF539619, LC339936, EU315064.1, and JF491202.1) from GenBank, but they were all A. solani of diferent strains with 97-100% similarity with those in GenBank. Reports of Mugao et al. [36] revealed that all the 19 isolates were A. solani with diferent accession numbers (LN879928, MN871610, MN871616, and MN871613) indicating the diferences in the genetic makeup. According to Loganathan et al. [37], the fve isolates of A. solani studied had the same genetic makeup with all of them possessing the same accession number (JF796068) from GenBank. Studies have shown that A. solani from the same lesions can be genetically distinct, indicating the high variability of the pathogen [32]. Tese genetic variations may be brought about by mutations, selection, and gene fow, and this infuences disease dynamics and fungicide resistance [44]. Reports of Leiminger et al. [48] showed that twenty isolates obtained from identical geographical sites formed diferent subgroups, having distant similarities that ranged between 75 and 95%. Van der Waals et al. [43] studied 46 isolates of A. solani and reported that 44 of them were unique genotypes and the gene diversity was 0.27. Taylor et al. [49] revealed a high genetic diversity of A. solani using VC and RAMS markers. McDonald and Linde [50] also reported high genetic diversity among A. solani isolates and concluded that it poses a high risk of breaking down genetic resistance in the host plants.
Isolates identifed as A. solani from potato and tomato host plants showed large genetic distances based on RAPD analysis in the United States [51] and by AFLP markers in Cuba [44]. Tiwari and Chitora [52] isolated DNA from 12 isolates, and gene sequencing alienated the isolates into three cluster groups. Group one contained 10 isolates, while groups two and three were made up of one isolate each. Similarly, reports of Upadhyay et al. [38] revealed that the gene sequence of 32 isolates of A. solani grouped them into two groups, but group one had two subgroups depending on similarity percentage from GenBank. In USA, high genetic diversity of isolates from tomato and potato plants was detected using isozymes [53].

Morphological and Molecular Diversity of
Phytophthora infestans 4.1. Morphological Diversity of P. infestans. P. infestans has white fufy mycelia with beige tones caused by the presence of sporangia with hyphae measuring 5-8 μm in diameter, hyaline and ramifed. Te mycelia were white in colour and aseptate with long sporangiophores, 5 μm pedicel, and ellipsoidal sporangia [54]. Reports of Dida et al. [55] showed a total of 8 shapes of sporangia in Cameroon. Tese shapes were as follows: oval to ellipsoid, ellipsoid, pip form, elliptic, subglobose, globose, ovoid, and lemoniform, implying diversity among them. Te sporangia length ranged between 0.63 μm and 0.96 μm, while the width ranged between 0.36 μm and 0.50 μm, respectively. Studies carried by [56] in Ethiopia revealed two distinct lineages. Te Ia mtDNA haplotype lineage had the sporangia that were ellipsoid to ovoid, caduceus, and semipapillate. According to Shimelash and Dessie [56], the sizes of sporangia from the studied P. infestans averaged 60.5 μm length and 31.7 μm width. Te sporangia were mostly ellipsoid to ovoid. Tey were caduceus and semipapillate for Ia mtDNA haplotype lineages of P. infestans and papillate for Ic mtDNA haplotype lineages of P. infestans [56]. Tis lineage also revealed the presence of chlamydospores, but all of them were of A1 mating type. Te Ic mtDNA haplotype lineage had sporangia that were ellipsoid to ovoid, caduceus, and papillate arranged in simple and sympodia sporangiophores. Te sizes of sporangia averaged 60.5 μm in length and 31.7 μm in width. Te Ic mtDNA haplotype lineages had chlamydospores at the terminal hyphae. Te two haplotype lineages were of A1 mating type.

Genetic Diversity of Phytophthora infestans.
Mitochondria and the DNA in the nucleus of P. infestans have been used to evaluate its population structure and evolutionary history, and there are two mating types that coexist and can reproduce and survive sexually [25]. Although P. infestans undergoes frequent genetic changes [57], the pathogen portrays low genetic diversity and its distribution and population structure is infuenced by the host preference [58]. Each host group of P. infestans is associated to diferent clonal lineage: tomato with US-1, potato with EC-1, wild solanaceous species with EC-2, and S. betaceum EC-3 [58]. P. infestans has been classifed into four main groups based on mitochondrial haplotypes: Ia, Ib, IIa, IIb [31], and 1c [59]. Te EC-2 and EC-3 lineages has been associated with the Ia haplotype, the US-1 lineage to the Ib haplotype, the US-6 to the IIb haplotype, the EC-1 to the IIa haplotype [31], and EC-2 has also been related to haplotype IC and correlated with new species, P. andina [59]. Hussain et al. [60] identifed two P. infestans mating types A1 and A2, and all had amplicons of 524 bp using SCAR markers. Reports of Arafa et al. [61] identifed P. infestans mating type A1 using SSR markers in Egypt with seven genotypes among fourty isolates indicating genetic variability. According to Santana et al. [62], 131 potato and 32 tomato isolates were evaluated where 59 isolates were classifed as A1 mating type, while 103 were A2. All the tomato isolates that were of A1 mating type presented 86/ 100 pattern for the enzyme GPI and mtDNA Ib, indicating that the isolates belonged to the US-1 clonal lineage. Of the 131 potato isolates, 103 were A2, while 27 were of A1 mating type. Among the potato isolates, 27 exhibited the Gpi phenotype 100/100, the same as BR-1 (mating type A2) and 20 were 86/100, the same as US-1 (Mating type A1). Studies conducted in Brazil revealed the presence of the two mating types of P. infestans [63]. Te US-1 lineage (mating type A1) is said to have many variants with at least 19 US genotypes [64].
In Russia, a study carried out by Beketova et al. [65] reported more (70%) A2 mating type than (30%) A1 in the study area. According to Cooke et al. [66], a clonal lineage of a new population known as 13_A2 which is also known as Blue-13 was discovered and was said to be more aggressive in Switzerland and Denmark. In UK and France clone, 6_A1 was discovered [66]. In Ireland genotype, 8_A1 of P. infestans was also discovered [67]. Te population of P. infestans in Ethiopia had been described in two studies where the majority of all the isolates were described as Ia mitochondrial DNA haplotype with A1 mating type [68]. In Canada and the United States, two major genotypes of the pathogen, an A2 mating type (US-8) and an A1 mating type (US-11), were discovered [69,70]. Te presence of US-14 genotype of P. infestans and another isolate with a unique fnger print were identifed in New Jersey indicating variability [70]. P. infestans population of A2 mating type (US-22) was also identifed in the United States and Canada [71]. Furthermore, clonal lineages US-23 and US-24 have also been identifed in the United States with some important diferences within the lineages [72].
Vargas et al. [73] conducted a study in Peru and Ecuador and found out that among the 64 isolates 59 were mating type A1, while the rest were mating type A2. PCR amplifcation performed by Trout et al. [74] on P. infestans collected from Tennessee, north and south Carolina, using PINF and ITS5 primers revealed band sizes of 600 bp. Sixtyfve samples collected from Jordan valley and analysed by Wesam et al. [75] through PCR amplifcation yielded 990 pb. Khalid et al. [76] amplifed the genomic DNA of P. infestans using ITS 3 and ITS 4 primers and yielded a band of 612 bp, while amplifcation with PINF and ITS 5 primers yielded a band of 600 pb [74]. Variations in band sizes indicate variability in their DNA.

Conclusion
Te knowledge of genetic diversity of plant pathogens is important in selection of resistance genes in breeding programs. Terefore, the use of decision support systems should be incorporated to the management of early blight and late blight on potato and tomato crops to optimize fungicide spraying, avoiding environmental contamination, and the selection of resistant isolates. Preventive control with protectant fungicides must be always conducted. Further studies are necessary to reveal the aggressiveness, distribution, symptoms' pattern in the feld, and diference in ftness of the species.

Conflicts of Interest
Te authors declare that they have no conficts of interest.