Sexual Compatibility Types in F1 Progenies of Sclerospora graminicola, the Causal Agent of Pearl Millet Downy Mildew

Sclerospora graminicola is primarily heterothallic in nature with two distinct mating types (G1 and G2); however, homothallism does exist in the pathogen populations. In this study, a cross was made between two self-sterile isolates (Sg 019, Mat-2, G2 × Sg 445-1, Mat-1, G1) of S. graminicola and a total of 39 F1 progenies were established. The study on sexual compatibility types in F1 progenies was conducted by crossing each F1 progeny with both the parents (Sg 445-1, Mat-1, G1; and Sg 019, Mat-2, G2). The results revealed the presence of four sexual compatibility types, viz. G1, G2, G1G2 and G0 (neuter) in the progenies. The G1G2 progenies that produced oospores with both the parents were found as self-fertile (homothallic) and self-sterile (heterothallic) types. Similarly, self-fertile parental type G1 and G2 progenies were designated as secondary homothallic, whereas self-sterile parental type G1 and G2 progenies were of heterothallic type. The result of the present study revealed Mendelian segregation of mating type locus in S. graminicola which indicates that sexual reproduction plays an important role in the evolution of new genetic recombinants in the pathogen. The study also helps in understanding the genetic structure of S. graminicola populations and potential for possible evolution of new virulences in the pathogen.


Introduction
Pearl millet [Pennisetum glaucum (L) R. Br.] is a choice crop of more than 90 million people cultivated on approximately 27 million hectares in the arid and semi-arid tropics of the world [1]. In India, mainly the states of Rajasthan, Gujarat, Haryana, Maharashtra, Uttar Pradesh, Karnataka and Andhra Pradesh produce 8.74 million tons of pearl millet. The crop is cultivated on 7.20 million hectares of land with a productivity of 1214 kg ha −1 [2]. Although average productivity of pearl millet in India has increased since the 1950s (305 kg ha −1 ) [3], it has also witnessed the devastating crop losses of up to 80% at periodic intervals caused by the downy mildew (DM) pathogen, Sclerospora graminicola [(Sacc). Schroet] [4]. The corresponding changes in the population structure of the pathogen over a period of time have played a key role in the destruction of the crop. The reason behind the evolution of new pathotype/s has been attributed to extreme selection pressure from the host along with sexual reproduction in S. graminicola populations [5].
The oospores formation in S. graminicola has been reported either through heterothallism, in which two self-sterile isolates having distinct sexual compatibility types, G 1 and G 2 , fuse together [6,7], or through secondary homothallism in self-fertile isolates that contain the determinant of both compatibility types [8]. In general, one isolate produces functional antheridia and the other isolate forms oogonia during a reciprocal crossing between two self-sterile isolates and the evidence of relative sexuality within isolates determines the contribution of antheridia and oogonia by each parent [9]. However, the presence of multiple compatibility types has been reported in other oomycetes. Four compatibility types (A 1 , A 2 , A 1 A 2 and neuter) have been observed in the F 1 progenies of the crosses derived from two distinct mating type isolates (A 1 × A 2 ) of Phytophthora spp. [10,11]. The production of oospores in one mating type (G 2 ) of S. graminicola isolate without fusion with any mating type [6] and no formation of oospores in isolate Sg 110-2 with any one of the designated mating types (G 1 and G 2 ) [12] indicated the presence of multiple compatibility types in S. graminicola [6]. Therefore, this study was planned to investigate the occurrence of self-sterile, self-fertile and neuter (sterile) isolates in S. graminicola to ascertain the multiple sexual compatibility types within the pathogen.

Collection and Maintenance of Isolates
A total of 52 isolates of S. graminicola were collected from different pearl millet growing areas of India during 1992 to 2012 ( Table 1). The single zoospore isolates of each collection were established [12] and were maintained separately either on their original host or on another susceptible host in the isolation polyacrylic chambers (60 cm × 45 cm × 45 cm) in the glasshouse at ICRISAT, India.

Identification of Self-Sterile or Self-Fertile Isolates
To identify the homothallic or heterothallic isolates, the single zoospore isolatesinfected plants were allowed to mature for formation of oospores in separate isolation chambers. Necrotic leaf pieces from 2-month-old seedlings infected with each isolate were collected in brown paper bags, cut into 1-centimeter-long pieces, dried under shade and stored at room temperature (25 ± 2 • C) until further observation. The small leaf pieces were surface sterilized with NaOCl (2%) and washed thoroughly with sterilized distilled water. These leaf pieces were cleared by incubating them at 40 • C in NaOH (5%) for 12 to 16 h. Cleared leaf pieces were rinsed in distilled water and observed under a microscope using a 10× objective for the presence of oospores. Isolates which did not show oospore formation were selected as self-sterile isolates for further studies.

Selection of Highly Virulent Self-Sterile Isolate
The sporangial inocula of all the self-sterile heterothallic isolates were raised on seedlings of a highly susceptible genotype 7042 S in isolation chambers in the glasshouse. The sporangia from sporulating leaves were harvested in ice-cold distilled sterile water and spore concentration was adjusted to 1 × 10 6 mL −1 . Pot-grown seedlings of the pearl millet differential lines P 7-4, P 310-17, 700651, 7042 R, IP 18292, IP 18293 and 852 B and two known downy mildew (DM) susceptible lines-ICMP 451 and 7042 S-were spray-inoculated at coleoptile stage using an atomizer. The inoculated seedlings were incubated at 20 • C with >90% Relative Humidity (RH) for 20 h, and then transferred to greenhouse benches at 25 ± 2 • C and >90% RH for disease development for the next 2 weeks. DM incidence was recorded 14 days after inoculation as percentage of infected plants. The isolates with ≤10% disease incidence were considered avirulent and those with >50% disease incidence as virulent on the specific genotype.

Confirmations of Mating Type of Virulent Test Isolate (Sg 445-1)
The reference isolates Sg 018 (Mat-1, G 1 ) and Sg 019 (Mat-2, G 2 ) and test isolate Sg 445-1 (single zoospore selection from Sg 445) of S. graminicola were maintained separately on 7042 S. To detect the mating type of the test isolate, Sg 445-1 was crossed with both the reference mating type isolates (Sg 018 × Sg 445-1; and Sg 019 × Sg 445-1). Sporangial inoculum of each isolate (1 × 10 6 sporangia mL −1 ) was prepared individually in ice-cold distilled sterile water. Sporangial suspensions of Sg 018 and Sg 445-1, and Sg 019 and Sg 445-1 were mixed in equal proportion (1:1) and spray inoculated on the highly susceptible pearl millet line 7042 S separately. The inoculated seedlings were incubated and transferred to isolation chambers. The infected seedlings were grown in the isolation chambers and allowed to mature. The necrotic tissues from these infected seedlings (>2 months old) were observed for oospore formation.

Establishment of F 1 Progenies from Oospores Generated from Sg 019 × Sg 445-1 Crosses
To generate progenies from F 1 oospores (Sg 019 × Sg 445-1), infected leaf samples with oospores were dried in the shade, grinded and strained to make a fine powder. Oospores were checked again for their presence in the matured leaf powder. Sterilized potting mixture (soil, sand, and farmyard manure in a ratio of 3:2:2 by volume) was infested with oospore inoculum (20-25 g) and the pots (15 cm diameter) containing the infested mixture were sown with a susceptible genotype 7042 S (25 seeds per pot). Each pot was covered with a polythene bag and incubated at 40 • C for 3-4 days for rapid seed germination. Pots were transferred to isolation chambers in a glasshouse at 25 ± 2 • C to avoid any cross contamination from other isolates. Pots were watered adequately every day and observed regularly for DM symptoms on the seedlings. When the first infected seedling in a pot was noticed, it was removed from the pot and was transplanted into another pot containing sterilized soil and shifted to an isolation chamber. Sporangia from each seedling were maintained separately on 7042 S as an individual F 1 -progeny in isolation chambers at 25 ± 2 • C in the glasshouse. A total of 39 F 1 progenies were established to determine sexual compatibility types in S. graminicola. Since infected seedlings occurred infrequently and rarely, each infected seedling was assumed to have infection from a single oospore.

Identification of Sexual Compatibility Types and Self-Sterile/Fertile Nature of F 1 Progenies
To detect sexual compatibility types of F 1 progenies, all the 39 F 1 progenies derived from the cross Sg 019 × Sg 445-1 were crossed with both the parents (Sg 445-1, Mat-1, G1; and Sg 019, Mat-2, G2) separately. Sporangial inoculum (1 × 10 6 sporangia mL −1 ) of each of the F 1 progenies and both the parents was prepared separately in ice-cold distilled sterile water, mixed in equal proportion (1:1) and spray inoculated on the highly susceptible pearl millet line 7042 S separately. The inoculated seedlings were incubated, transferred to isolation chambers and the infected seedlings were allowed to mature for production of oospores. In addition, to identifying the self-sterile or self-fertile nature of F 1 progenies, the single-zoospore infected plants were allowed to mature in separate isolation chambers and observed for the presence of oospores.

Selection of Self-Sterile Heterothallic Isolates
The 60-day-old, infected leaves of 52 single-zoosporic isolates of S. graminicola were checked for presence of oospores. No oospores were detected in 33 isolates, whereas oospores were formed by the remaining 19 isolates (Table 2). Isolates without oospores formation were designated as self-sterile or heterothallic while those producing oospores were designated as self-fertile or homothallic. Thus, a total of 33 heterothallic isolates were selected and the 19 homothallic isolates were excluded from the further studies.

Selection of Highly Virulent Self-Sterile Isolate
All the 33 self-sterile heterothallic isolates including reference mating type isolates Sg 018 (Mat-1/G 1 ) and Sg 019 (Mat-2/G 2 ) were screened on seven host differentials (P 7-4, P 310-17, 700651, 7042 R, IP 18292, IP 18293 and 852 B) and the two known DM susceptible lines (ICMP 451 and 7042 S). The screening identified Sg 445-1 as the most virulent isolate and Sg 018 and Sg 019, the two reference mating type isolates, as avirulent on specific genotypes; hence, they were selected for the crossing and generation of F 1 progenies (Table 3).
Thus, two parents Sg 019 (avirulent) and Sg 445-1 (virulent) of different mating types were selected for crossing and generation of 39 F 1 progenies.

Identification of Sexual Compatibility Types and Self-Sterile/Fertile Nature of F1 Progenies
A total of 39 F 1 progenies were derived from the cross of Sg 019 Mat-2, G 2 × Sg 445-1 Mat-1, G 1 . In contrast to the distinct mating types of the parents (G 1 and G 2 ), progenies were of four compatibility types viz. G 1 , G 2 , G 1 G 2 and G 0 (neuter) ( Table 4). Of 39 F 1 progenies, four belonged to G 1 , 13 to G 2 , 21 G 1 G 2 and one to neuter categories (Tables 4 and 5). Further, the self-fertile or self-sterile nature of all the 39 F 1 progenies was evaluated on the basis of production of oospores. Among 21 G 1 G 2 progenies, 19 supported self-production of oospores while 2 were free of any oospores in the matured leaves. Out of four G 1 progenies, oospores were observed in three progenies and one was recorded as a non-oospore producer when selfed. Of the 13 G 2 progenies, 7 supported self-production of oospores whereas no oospore formation was observed in the matured leaves infected with the remaining 6 F 1 progenies. Table 4. Determination of sexual compatibility types of F 1 progenies based on oospores formation with Sg 445, Mat-1 (G 1 ) and Sg 019, Mat-2 (G 2 ).

Population
Oospore Formation with Mating Type of Population Self-Fertile/ Sterile Remarks Sg 445-1 (G 1 ) Sg 019 (G 2 ) One unique neuter (G 0 ) progeny was recorded as a non-oospore former, which was neither self-fertile nor produced oospore by crossing with any of the two parents. The F 1 progenies which produced oospore by crossing with both the parents were designated as G 1 G 2. Both self-sterile and self-fertile progenies were observed among G 1 G 2 s. In S. graminicola, it is reported that oospore formation is very low when isolates are selfed, whereas the number of oospores formed is quite high when the isolates of different mating types are crossed [6,12]. Similar observations were made in the present study. In the case of selfed G 1 G 2 F 1 s, about 10 oospores were observed per leaf piece (1 cm 2 ), whereas 100-300 oospores were found when they were crossed with either of the parents. Thus, the 19 self-fertile (G 1 G 2 ) progenies, which showed production of oospores with both parents, were designated as homothallic, while two self-sterile (G 1 G 2 ) progenies were designated as heterothallic type (Table 5). Similarly, the self-fertile parental type G 1 and G 2 proge-nies were denoted as secondary homothallic whereas self-sterile parental type G 1 and G 2 progenies were of heterothallic type. Table 5. Summary of determination of sexual compatibility types of F 1 populations based on oospore formation with Sg 445, Mat-1(G 1 ) and Sg 019, Mat-2 (G 2 ).

Discussion
The oospore formation in plant pathogenic oomycetes depends on the presence of two sexual compatibility types or their determinants [13][14][15][16][17]. In S. graminicola, two types of mating/compatibility types, viz. G 1 and G 2 , have been proposed earlier [6,7,12] which are responsible for sexual reproduction between two self-sterile isolates, and within self-fertile isolates. Since sexual reproduction is dependent upon both compatibility types, it is speculated that the self-fertile isolates contain both compatibility types in the same seedling. The earlier studies [6,7,12] also reported self-fertile isolates and placed these isolates in G 2 mating types tentatively and suggested that determination of sexual compatibility type in S. graminicola is likely to be complex and the nomenclature of G 1 /G 2 compatibility types may not necessarily imply their distribution in a population. In addition, the neuter (sterile) type of S. graminicola isolate (Sg 110-2) was also observed [12], which failed to produce oospores with any of the parent isolates and was also placed under G 1 /G 2 compatibility types.
Since vegetative structures of oomycetes exist in diploidy level, the mating type alleles have been reported to be controlled by a single mating type locus in Phytophthora spp. [11,18,19] due to equal numbers of A 1 or A 2 types in the progenies. However, skewed numbers of one or the other mating types have also been reported [10,[20][21][22]. Although normal Mendelian segregation of alleles expects four different combinations of alleles for a given locus in the progenies of heterozygous parents, inheritance of mating type alleles of a single locus has been explained in three different ways to explicate the almost equal ratios of A 1 and A 2 progenies in Phytophthora spp. [11,18,20].
In the first model, one mating type is represented by heterozygous (A/a) condition and the other in homozygous (a/a) condition at the mating type locus [20] which can yield only two types of sexual compatibility types in the offspring. However, inconsistent ratios in the progenies of heterozygous (A/a) and homozygous (a/a) parents have been reported in contrast to this model [15,22,23]. The second model suggests the presence of balanced lethal loci due to survival of only two genotypes A 1 (M 1 /M n ) and A 2 (M 2 /M n ) instead of the four different genotypes (M 1 /M n , M 2 /M n , M 1 /M 2 or M n M n ) in the progenies of A 1 (M 1 /M n ) and A 2 (M 2 /M n ) mating type parents in Phytophthora infestans [18]. The third model, a hybrid of the earlier two, explains the existence of ambiguous A 1 -A 2 genotype in P. parasitica, which was consistent with the first model in which the A 1 mating type was represented by heterozygous (M A /M a ) and A 2 in homozygous (M a /M a ) conditions for the alleles at the mating-type locus [11]. In contrary to all three models, the present study revealed four different compatibility types (4G 1 , 13G 2 , 21G 1 G 2 and one G 0 , neuter) in 39 F 1 progenies from the cross of two distinct self-sterile heterothallic parents (Sg 445-1 Mat-1, G 1 × Sg 019 Mat-2, G 2 ) that indicated normal Mendelian segregation of mating types (Table 6) in S. graminicola. In the earlier studies [6,12], four different compatibility types were also noticed in S. graminicola though all the progenies were accommodated in G 1 /G 2 compatibility types either due to skewed distribution of mating types or lack of nomenclature in S. graminicola. The discussed three models were found inadequate to explain the usual segregation in S. graminicola and unequal ratio of G 1 :G 2 along with ambiguous G 1 G 2 sexual compatibility types. Therefore, an alternative scheme for matingtype determination was considered and the segregation could be speculated due to presence of mating type alleles in heterozygous state in both parents [G 1 g 1 (Mat-1) for G 1 and G 2 g 2 (Mat-2) for G 2 ] at the same locus. In Phytophthora, isolates forming oospores only with the A 1 or A 2 testers are designated as A 2 and A 1 , respectively, whereas the isolates which can form oospores with both A 1 and A 2 testers are designated as A 1 A 2 and those that fail to form oospores are designated as A 0 (sterile or neuter) [24] which supports the results of this study. Table 6. Mendelian segregation of sexual compatibility types in two distinct self-sterile heterothallic parents (Sg 445-1, Mat-1, G 1 × Sg 019, Mat-2, G 2 ) of Sclerospora graminicola. and A2 (M2/Mn) mating type parents in Phytophthora infestans [18]. The third model, a brid of the earlier two, explains the existence of ambiguous A1-A2 genotype in P. parasi which was consistent with the first model in which the A1 mating type was represen by heterozygous (MA/Ma) and A2 in homozygous (Ma/Ma) conditions for the alleles at mating-type locus [11]. In contrary to all three models, the present study revealed f different compatibility types (4G1, 13G2, 21G1G2 and one G0, neuter) in 39 F1 proge from the cross of two distinct self-sterile heterothallic parents (Sg 445-1 Mat-1, G1 × Sg Mat-2, G2) that indicated normal Mendelian segregation of mating types (Table 6) i graminicola. In the earlier studies [6,12], four different compatibility types were also ticed in S. graminicola though all the progenies were accommodated in G1/G2 compatib types either due to skewed distribution of mating types or lack of nomenclature in S. gr inicola. The discussed three models were found inadequate to explain the usual segre tion in S. graminicola and unequal ratio of G1:G2 along with ambiguous G1G2 sexual c patibility types. Therefore, an alternative scheme for mating-type determination was c sidered and the segregation could be speculated due to presence of mating type allele heterozygous state in both parents [G1g1 (Mat-1) for G1 and G2g2 (Mat-2) for G2] at the sa locus. In Phytophthora, isolates forming oospores only with the A1 or A2 testers are de nated as A2 and A1, respectively, whereas the isolates which can form oospores with b A1 and A2 testers are designated as A1A2 and those that fail to form oospores are de nated as A0 (sterile or neuter) [24] which supports the results of this study. Table 6. Mendelian segregation of sexual compatibility types in two distinct self-sterile heteroth parents (Sg 445-1, Mat-1, G1 × Sg 019, Mat-2, G2) of Sclerospora graminicola.
G2 g1 (Mat-2) g1 g2 (G0, Neuter) The mating system plays an important role in the evolution of plant pathogens d ing strong selection pressure from the resistant host or chemical control measures or ha environmental conditions [25,26]. In oomycetes, the predominant co-existence of two m ing types (G1 and G2 or A1 and A2) [6,7,11,12,18,19] and generation of multiple compat ity types (A1, A2, A1A2 and neuter) in the F1 progenies upon sexual reproduction betw two distinct mating types (A1 × A2) [10,11] might provide advantage to pathogens dur G 2 g 2 four different genotypes (M1/Mn, M2/Mn, M1/M2 or MnMn) in the progenies of A1 (M1/Mn and A2 (M2/Mn) mating type parents in Phytophthora infestans [18]. The third model, a hy brid of the earlier two, explains the existence of ambiguous A1-A2 genotype in P. parasitica which was consistent with the first model in which the A1 mating type was represented by heterozygous (MA/Ma) and A2 in homozygous (Ma/Ma) conditions for the alleles at th mating-type locus [11]. In contrary to all three models, the present study revealed fou different compatibility types (4G1, 13G2, 21G1G2 and one G0, neuter) in 39 F1 progenie from the cross of two distinct self-sterile heterothallic parents (Sg 445-1 Mat-1, G1 × Sg 01 Mat-2, G2) that indicated normal Mendelian segregation of mating types (Table 6) in S graminicola. In the earlier studies [6,12], four different compatibility types were also no ticed in S. graminicola though all the progenies were accommodated in G1/G2 compatibility types either due to skewed distribution of mating types or lack of nomenclature in S. gram inicola. The discussed three models were found inadequate to explain the usual segrega tion in S. graminicola and unequal ratio of G1:G2 along with ambiguous G1G2 sexual com patibility types. Therefore, an alternative scheme for mating-type determination was con sidered and the segregation could be speculated due to presence of mating type alleles in heterozygous state in both parents [G1g1 (Mat-1) for G1 and G2g2 (Mat-2) for G2] at the sam locus. In Phytophthora, isolates forming oospores only with the A1 or A2 testers are desig nated as A2 and A1, respectively, whereas the isolates which can form oospores with both A1 and A2 testers are designated as A1A2 and those that fail to form oospores are desig nated as A0 (sterile or neuter) [24] which supports the results of this study.