Metalaxyl-resistant mutant strains of Phytophthora boehmeriae are as aggressive and fit as their metalaxyl-sensitive wild-type parents

Phytophthora boehmeriae Sawada is an important pathogenic oomycete, and causes Phytophthora blight of cotton and ramie, which are main limiting factors to the production of cotton and ramie in China. The aggressiveness and fitness of the metalaxyl-resistant (MR) mutants of P. boehmeriae obtained by inducing on LBA amended with sublethal dose (10 µg·mL−1) of metalaxyl were studied in this paper. The results showed that there were no significant differences between the MR mutants and their metalaxyl-sensitive (MS) wild-type parents in temperature for mycelial growth, zoosporangium production, sensitivity to malachite green, and the pathogenicity to cotton seedlings. However, the oospore productions of the mutants were much lower than those of their MS parents. The growth rates and the colony morphology of MR mutants and their MS parents all displayed obvious variation or separation, and were inherited unsteadily in their single-zoospore and single-oospore progenies. At the same time, the homothallic character of MR mutants could inherit steadily as well as that of the MS wild-type isolates did in asexual and sexual progenies. It was suggested that the MR mutants of P. boehmeriae possessed equal aggressiveness and fitness with their MS parental isolates, and would develop the MR population easily in a short time as long as the MR mutants formed. Thereby, there would be a high risk of development of metalaxyl resistance in populations of P. boehmeriae in the field, when metalaxyl was long and continuously applied for the control of the diseases caused by P. boehmeriae.


Introduction
The oomycetes in Phytophthora are important plant pathogens and are widely distributed around the world (Erwin and Ribeiro 1996;Li et al. 2007;Stukely et al. 2007;Burgess et al. 2009;Hughes et al. 2011;Yang et al. 2011;Wang et al. 2020a). Phytophthora blight caused by Phytophthora boehmeriae Saw. is a limiting factor for the production of cotton, ramie, citrus, black wattle, chili, and several other plants (D'Souza et al. 1997;Ho and Lu 1997;Elena and Paplomatas 1998;Thomidis et al. 2002;Dos Santos et al. 2006;Wu et al. 2010;Chowdappa et al. 2014;Alves et al. 2019; Thorpe et al. 2021). In China, P. boehmeriae is the causal organism of cotton blight and ramie blight, causing destructive disease losses frequently (Ho and Lu, 1997;Chen et al. 2001;Gao 2007;Wu et al. 2010;Wang et al. 2020a). Metalaxyl, a systematic phenylamide fungicide, has excellent protective, curative, and eradicative activity against oomycetes, and is widely used to control the plant diseases caused by oomycete pathogens, including cotton blight (Bruin and Edgington 1982;Gisi and Cohen 1996;Belisle et al. 2019). Application methods for disease control include seed treatment, foliar spray, and soil drench. For the control of cotton blight, fungicides can be sprayed in seedling stage for the control of seedling blight and in the boll-bearing period for the control of boll blight (Gao 2007). As metalaxyl is a single-site specific fungicide with the action mode of selective inhibition of ribosomal RNA synthesis by affecting the activity of RNA polymerases, the pathogenic oomycetes develop resistance to the fungicide easily after its application (Davidse et al. 1983;Gisi and Sierotzki 2008;Randall et al. 2014). For this reason, Fangxin Chen and Yongxia Qi contributed  and due to selection pressure brought by a long use of the fungicide, developments of resistance to metalaxyl among members of the oomycetes, including the genus Phytophthora, have appeared in many countries (Café-Filho and Ristaino 2008;Gisi and Sierotzki 2008;Hu et al. 2008;Pérez et al. 2009;Lookabaugh et al. 2018;Gray et al. 2020;Gonzalez-Tobon et al. 2022).
Resistance to metalaxyl of P. boehmeriae was first demonstrated experimentally in the laboratory by Gao et al (1997). The authors reported that the metalaxyl-resistant (MR) mutants were obtained from wild-type metalaxylsensitive (MS) isolates of P. boehmeriae by inducing on LBA amended with sublethal dose of metalaxyl. The resistance level of the MR mutants was over 1800 times higher than that of the wild-type MS parent isolate. The resistance of some mutants to metalaxyl could steadily inherit in their asexual and sexual generations, but the others not (Chen et al. 2004a). The results suggested that there was a risk of development of metalaxyl resistance in populations of P. boehmeriae under field situations. However, metalaxyl is still one of the most popular fungicides for the control of oomycetes, and is still recommended and used in China for control of plant diseases caused by Phytophthora species. Moreover, the fitness and stability of MR isolates of P. boehmeriae are unknown to date.
Fitness is defined as an organism's ability to contribute to the subsequent gene pool, and its main components include growth rate, reproductive ability, and pathogenicity/aggressiveness (Hu et al. 2008). There were some reports on the fitness of MR isolates in other species of Phytophthora. Bruin and Edington (1982) found that the MR mutants of P. capsici generally grew much more slowly than the parent isolates on unamended V8 juice agar, suggesting reduced fitness in the absence of selection pressure from the fungicide. In addition, some of the MR mutants reverted to sensitivity after successive transfers on unamended media, indicating that MR mutants were less fit than the wild types. On the other hand, Bower and Coffey (1985) induced metalaxyl resistance in P. capsici by chemical mutagenesis and found that most resistant isolates remained resistant to metalaxyl without any loss of pathogenicity after many transfers in the absence of selection pressure on unamended cornmeal agar. Café-Filho and Ristaino (2008) reported that MR isolates of P. capsici are as virulent and fit as MS isolates: when measured by rate of colony growth, sporulation in vitro, and aggressiveness in planta, fitness of MR isolates was not reduced, and MR isolates were as aggressive on pepper as MS isolates. Our lab (Qi et al. 2011;Wang et al. 2020bWang et al. , 2021 also demonstrated that there were no significant differences between MR isolates and MS isolates of P. capsici in radical growth rate and pathogenicity to pepper. Bashan et al. (1989) found that in the absence of metalaxyl, MR isolates of P. infestans were more infective to potato foliage than MS isolates during relatively short periods of leaf wetness. Stack and Millar (1985) described that a MR isolate (Pm20) of P. megasperma f. sp. medicaginis did not differ from the wild type in growth rate, propagule germination, and pathogenicity to alfalfa. Grinberger et al. (1995) observed that MR isolates of P. infestans were not different significantly from sensitive isolates in percent of infected tubers, and produced larger and deeper lesions compared to MS isolates, and concluded that the resistant isolates cause more severe tuber blight. Gisi and Cohen (1996) discovered that MR isolates of P. infestans survived poorly in infected potato tubers compared with MS isolates, but during the growing season, especially in the case of application of metalaxyl, sporulation capacity of MR isolates was stronger than that of MS isolates. Moreover, MR isolates of P. infestans (Kadish and Cohen 1988) and P. nicotianae (Timmer et al. 1998;Hu et al. 2008) in the field were more aggressive than their MS counterparts.
However, the biological characteristics and their inheritance of the MR mutants of P. boehmeriae had not been reported, and the relative fitness of MR mutants of the oomycete is unknown to date. In other words, we have not known that whether the MR mutants or isolates of P. boehmeriae are as aggressive and fit as their MS progenitors or not, which is very important for the risk evaluation and management of metalaxyl resistance of P. boehmeriae. Therefore, in the present study, the biological characteristics of the MR mutants of P. boehmeriae including zoosporangium and oospore production, mycelial growth rate, colony morphology, pathogenicity to cotton seedlings, homothallic character, and their genetic stability in asexual and sexual progenies were studied. The objective of this study was to evaluate the fitness and ability of MR isolates of P. boehmeriae and to provide a theoretical basis and experimental evidence for the risk assessment and management of metalaxyl resistance of P. boehmeriae.

Isolates tested
Isolates XC-6, WH-2, SX-9, WW-13, and WJ-5 of P. boehmeriae were isolated from diseased cotton rotten bolls collected in Xuancheng, Wuhu, Wuwei, and Wangjiang of China, respectively. Host tissue was surface-disinfected with 0.05% sodium hypochlorite for 1 min, and isolations were made on lima bean agar medium (LBA: lima bean powder 60 g, H 2 O 1000 mL, agar 18 g) amended with hymexazol (50 μg·mL −1 ). Petri dishes were incubated for 3 days in the dark at 25 °C, and colonies with characteristics of P. boehmeriae were transferred to 10% V8 juice agar (clarified V8 juice 100 mL, CaCO 3 2 g, deionized water 900 mL, and agar 18 g). V8 juice was clarified by filtration through a Whatman No. 4 filter after the addition of 2 g of CaCO 3 , followed by centrifugation at 5000 rpm for 10 min. The pathogen was identified based on colony characteristics on CMA (Gao et al. 1998a), sporangial morphology on 10% V8 juice agar, and by polymerase chain reaction (PCR) with the universal primers DC6 and ITS4 (Shen et al. 2005) following DNA extraction with a fast procedure (Zhang et al. 2006). All above isolates were sensitive to metalaxyl (the minimal inhibited concentration of mycelial growth, MIC＜1 µg·mL −1 ) by the metalaxyl sensitivity test (Gao et al. 1997), and reserved in the Fungal Lab of Anhui Agricultural University. Furthermore, the single-spore strains of these MS wild-type isolates were established by means of the single-spore isolation (Zheng 1997) for the mutagenesis experiments.

Fungicide
Metalaxyl (technical grade, 98%; Jiangsu Baoling Chemical Co., Ltd) was first dissolved in a small amount of acetone, and then diluted in sterilized distill water to stock solution at concentration of 5000 µg·mL −1 and stored at 4 °C.

Mutagenesis of metalaxyl-resistant mutants
To induce P. boehmeriae resistance against metalaxyl, the mutagenesis experiments were conducted by applying of the mycelial block induction with sublethal dose of metalaxyl (Gao et al 1997;Chen et al 2004a;Wang et al. 2020b). The MS P. boehmeriae isolates XC-6, WH-2, SX-9, WW-13, and WJ-5 were used as the wild-type strains. After the strains were cultured on LBA plates for 5 days, the 10 mm × 10 mm × 3 mm mycelium agar blocks were transferred onto LBA containing 10 µg·mL −1 metalaxyl, with four blocks on a Petri dish (9 cm in diameter) and 10 dishes for each isolate. The dishes were sealed with a strip of parafilm and incubated at 25 °C in the dark. After 7 days of incubation, the growth of the colonies was observed. After 10 to 14 days of incubation, if a rapidly growing sector (mycelial growth rate > 5 mm·day −1 ) appeared, the colonies in that sector were transferred onto LBA containing 10 µg·mL −1 metalaxyl. A normal mycelial growth rate (3-5 mm·day −1 ) was considered to be a sign of acquisition of metalaxyl resistance and such strains were chosen for further checkups. If such a strain could regularly grow on LBA containing 10 µg·mL −1 metalaxyl after being subcultured on LBA metalaxyl-free for over 3 times, it was identified as a MR mutant. The MR mutants were numbered according to their original isolates for further study.

Assessment of sensitivity to metalaxyl
Experiments to test the sensitivity of the mutants to metalaxyl were done within a month after isolation. Sensitivity to metalaxyl was estimated by measuring the radial colony growth of individual isolates on replicated plates of 10% V8 juice agar amended with metalaxyl at concentrations of 0, 5, and 100 µg·mL −1 with reference to Café-Filho and Ristaino (2008) with minor amendments. Mycelium agar discs (5 mm in diameter) from active growth zones of 3-day-old cultures of the tested isolates were transferred to each of three plates of media at each level of fungicide. Plates were incubated in the light at 25 °C for 3 days. Isolates were classified as sensitive, intermediate, or resistant based on their colony growth in the fungicide-amended media, relative to their respective growth on unamended 10% V8 juice agar. Isolates were characterized as sensitive if colony growth on media amended with 5 µg·mL −1 metalaxyl was less than 40% of that on unamended media. Intermediate isolates exhibited growth on media amended with 5 µg·mL −1 greater than 40% of that on unamended media, but growth on media amended with 100 µg·mL −1 less than 40% of that on unamended media. Resistant isolates exhibited growth on media amended with 100 µg·mL −1 greater than 40% of that on unamended media. The experiment was repeated twice.

Tests of mycelial growth rate and temperature tolerance
All tested MR mutants and their parental isolates were cultured on LBA for 3 days. A plug of mycelium (5 mm in diameter) from zones of active growth of 3-day-old culture was placed at the center of Petri dish (9 cm in diameter) for each temperature treatment. Three replicates for each temperature treatment were used. The dishes were incubated in the dark at 4 °C, 8 °C, 10 °C, 12 °C, 22 °C, 25 °C, 28 °C, 30 °C, 33 °C, 35 °C, and 36 °C for 3 days, respectively. Mean colony radius was measured for each plate to evaluate the growth. The whole trial was repeated twice.

Test of sensitivity to malachite green
Malachite green (AR, Lanyang Chemical Co., Ltd.) was dissolved in sterile water and diluted to final concentration of 0.0625, 0.125, 0.25, 0.5, 1.0, and 4.0 µg·mL −1 in LBA, respectively. A mycelial plug (5 mm in diameter) cut from zones of active growth of 3-day-old cultures was transferred to the center of each plate (9-cm-diameter Petri dish) with and without malachite green. Three replicates were used for each concentration treatment. Plates were placed randomly in an incubator and incubated in the dark at 25 °C for 72 h. Mean colony radius was measured for each plate to evaluate the growth. The whole trial was repeated twice.

Zoosporangium production determination
The determination of zoosporangium production was carried out with reference to Gao et al. (1997). Test isolates were grown on LBA plates in the dark at 25 °C. After 3 days, three 7-mmdiameter plugs of mycelium were cut from the growing edge of the colonies and transferred to 250-mL Erlenmeyer flasks containing 20 mL autoclaved 10% V8-broth (clarified V8 juice 100 mL, CaCO 3 2 g, deionized water 900 mL) amended with 10 μg·mL −1 metalaxyl and metalaxyl-free, respectively. After these Erlenmeyer flasks were incubated in the dark at 25 °C for 72 h, the mycelial blocks were transferred to 20 mL of sterile distilled water in Erlenmeyer flasks amended with 10 μg·mL −1 metalaxyl and metalaxyl-free, and incubated at 25 °C in the light for 48 h. During the incubating, the mycelial blocks were rinsed once every 12 h with sterile distilled water to induce sporangium production, and sporangial formation occurred after 36 to 48 h. Then, the mycelial block containing sporangia from each Erlenmeyer flasks, broth, and 20 mL distilled water was triturated in a mixer at 3000 rpm for 1 min. Subsequently, suspensions were constant volume to 50 mL with distilled water. For sporangium production, three replicates were used for each isolate combination, and the experiments were conducted twice. Numbers of sporangia were determined by counting the number of sporangia in 200-µL suspension with a hemocytometer, and sporangium morphology of the MR mutants and their parental strains were observed under a microscope.

Oospore production determination
A cork borer was used to take 7-mm-diameter discs of mycelium from the margin of actively growing colonies of 3-day-old cultures. Each mycelium disc was placed in the center of LBA plate (9 cm in diameter) amended with 10 µg·mL −1 metalaxyl and without metalaxyl, respectively. Ten replicates were for each of the isolates, and all the dishes were sealed with a strip of parafilm and incubated in the dark at 25 °C for 36 days. Twelve plugs of mycelium (7 mm in diameter) cut from the four corners of the square (40 mm × 40 mm) with the inoculation point as the center were transferred to a mixer, added 30 mL distilled water, and were triturated at 3000 rpm for 1 min. After that, suspension was diluted to 50 mL in volume with distilled water. Oospore production was determined by counting the number of oospores in 200-µL suspension with a hemocytometer, and the oospore morphology of MR mutants was observed (Gao et al. 1998a, b). The experiment was conducted three times.

Observation of mycelial growth rate, colony morphology, and homothallic character
By means of Gao et al.'s method (Gao et al. 1997), the obtained mutants, XC-6-2, XC-6-3, and WH-2-2, were used as the parents to establish their single-zoospore progeny and singleoospore progeny groups, respectively. Each single-spore isolate was cultured on LBA for 3 days at 25 °C. A plug of mycelium agar (5 mm in diameter) then from the growing edge of the culture was transferred to the LBA plate contained 20 mL medium and was incubated in the dark at 25 °C for 72 h. The colony radius was measured and colony morphology was observed for each plate. And 7 days after inoculation, the colony was observed five microscopic views (10 × 15) randomly to determine whether it had produced oospores or not. The MR mutants and the parental isolates were used as controls. Three replicates were applied for each treatment. Each strain was continuously tested for 3 to 4 generations. The whole experiment was conducted three times.

Experiment of pathogenicity to cotton seedlings
The experiments were conducted with reference to Gao et al. (1998b). Tested cotton cultivar was Simian 3. Cottonseeds were disinfected by concentrated sulfuric acid, and then were soaked in 55 to 60 °C hot water for 20 min. Subsequently, they were germinated for 24 h at 30 °C. Cotton seedlings were grown in the greenhouse at 10 days after sowing. The healthy cotton seedlings with the approximately same size were selected for inoculation tests when the cotyledons were fully unfolded. Tested isolates were grown on LBA plates in the dark at 25 °C for 3 days. Fivemm-diameter discs of mycelium agar were removed from the margin of the colonies and placed on the wound punctured with a bundle of 6 insect needles in the center of cotton cotyledons, and the control was inoculated with 5-mm-diameter LBA discs. The inoculated sites of cotton seedlings were covered with absorbent cotton dipped in sterile water for moistening, and incubated in the light at 25 °C. After 24 h, the discs and absorbent cotton were removed. The lesion diameter was measured after 48 h and 72 h. Six cotyledons were inoculated for each strain with three replicates. The aggressiveness /pathogenicity was estimated by the average lesion diameter of 6 cotyledons.

Statistical analysis of obtained data
DPS 7.0.5.8 software was applied for calculating the colony diameters, lesion expanding rates, sporangium production, oospore number and mycelial growth rates, and for the significance test (t-test) or variance analysis (F-test) of the characters tested above.

Growth temperature and linear rates of MR mutants of P. boehmeriae
The results of temperature experiment showed that the optimum temperature for mycelial growth of both MR mutants and their wild-type parents was ranging from 25 to 30 °C (Fig. 2). The highest growth temperature was less than 35 °C, and when the temperature was below 8 °C, the mycelia stopped growing. There were similar in tolerance to low temperature between the MR mutants and their wild-type parents. However, the mycelial growths of the MR mutants were faster compared with the parental at a higher temperature, which suggesting that mutation of P. boehmeriae in metalaxyl resistance did not change its adaptive range to temperature, and the MR mutants were equal in mycelium growth with their MS wild-type parents.

Sensitivity to malachite green of MR mutants of P. boehmeriae
The test result showed that the MR mutants and their parental isolates were all sensitive to malachite green (Table 2). There were weak growth on LBA amended with concentration of malachite green at 0.5 μg·mL −1 , but the growth was completely inhibited at the concentration of 1 μg·mL −1 . It was suggested that the MR mutants of P. boehmeriae and their parental were equal in malachite green sensitivity with their MS parents.

Aggressiveness of MR mutants of P. boehmeriae
The results of aggressiveness/pathogenicity to cotton seedlings showed that there were no significant differences in Fig. 1 The rapidly growing sectors appeared on some of the mycelia blocks of the MS wild-type isolates after 12 days of induction on the LBA amended with sublethal dose (10 µg·mL. −1 ) of metalaxyl. Left, a rapidly growing sector appeared on the mycelia block of isolate XC-6; right, a rapidly growing sector appeared on the mycelia block of isolate SX-9 Shouxian Isolated from diseased cotton boll S SX-9-1 -Obtained by metalaxyl induction from SX-9 R SX-9-2 -Obtained by metalaxyl induction from SX-9 R SX-9-4 -Obtained by metalaxyl induction from SX-9 R WW-13 Wuwei Isolated from diseased cotton boll S WW-13-1 -Obtained by metalaxyl induction from WW-13 R WW-13-3 -Obtained by metalaxyl induction from WW-13 R WJ-5 Wangjiang Isolated from diseased cotton boll S WJ-5-2 -Obtained by metalaxyl induction from WJ-5 R the average lesion expanding rates on cotton seedling leaves between the MR mutants of P. boehmeriae and their parental isolates (Table 3), which suggested that the MR mutants of P. boehmeriae possessed the equal aggressiveness to cotton seedlings with their parental isolates.

Sporangium production of MR mutants of P. boehmeriae
Productions of sporangia in 10% V8 broth were measured for each of MR mutants and their parental isolates. The results showed that there was no significant difference in the number of sporangia between each MR mutant of P. boehmeriae and its corresponding parental isolate, although there were significant differences among different MR mutant strains (Table 4). It was suggested that MR mutants of P. boehmeriae possessed equal ability of sporangial production with their corresponding parental isolates. On the other hand, the mycelial growth of MS isolates XC-6, WH-2 WW-13, and SX-9 was completely inhibited in 10% V8 broth amended with metalaxyl at the concentration of 10 μg·mL −1 , but MR mutants grew normally. It was also observed that the parental isolates did not formed sporangia in 10% V8 broth with metalaxyl at 10 μg·mL −1 , while the MR mutants formed sporangia easily, and the sporangia released zoospores normally. The sporangia produced by MR mutants had no obvious differences in morphology. It was suggested that metalaxyl could completely inhibit the mycelial growth and sporangium production of MS parental isolates with metalaxyl treatment of 10 μg·mL −1 , but had no obvious inhibition on MR mutants in mycelial growth, sporangium production, and release in the same situation.  XC-6-2, WJ-5-2, and SX-9-2 are the obtained MR mutant strains. XC-6, WJ-5, and SX-9 are MS wildtype isolates of P. boehmeriae, and used as the parental control isolates. The data are mean ± SE. Data in the same column followed by different lowercase and capital letters are significantly different at α = 0.05 and α = 0.01 level by Duncan's LSR test; " + " stands for weak growth, and " − " for no growth 4.28 ± 0.10 bAB 3.44 ± 0.10 abcAB 2.61 ± 0.10 abAB + + − SX-9-2 3.83 ± 0.00 cB 3.11 ± 0.10 cB 2.39 ± 0.10 bcABC + + − 1 3

Oospore production of MR mutants of P. boehmeriae
The oospore productions were determined on LBA in this experiment with the method mentioned above. The results showed that there were significant differences in oospore production between MR mutants XC-6-2, XC-6-3, and WH-2-2 and their corresponding wild-type parents, in which the oospore production of the three mutants was much lower than that of their parents (Table 5). However, MR mutant SX-9-2 was not significantly different from its wild-type parent SX-9. It was also observed that the parental isolates did not grew and formed oospores at all on LBA amended with metalaxyl at 10 μg·mL −1 , while the MR mutants formed oospores easily in the same experimental conditions. But the oospore production of MR mutants on LBA amended with metalaxyl at the concentration of 10 μg·mL −1 was obviously reduced, compared with metalaxyl-free treatment.

Mycelium growth rates
The genetic stability of the biological characters of MR mutants of P. boehmeriae was investigated. The results ( Table 6) indicated that there were significant differences in mycelium growth rates in the first single-zoospore generation (ZG 1 ) of MR mutants XC-6-2 and XC-6-3 and their parents. In the population of the second single-zoospore generation (ZG 2 ) established with the first single-zoospore generation (ZG 1 ) of isolate XC-6-2 as the parent, there were also significant differences in the growth rates among single-zoospore isolates. It was suggested that the growth rate character of MR mutants displayed sustaining variation. However, the growth rates of MR mutant groups of the second single-zoospore generation (ZG 2 ) established by the first single-zoospore generation (ZG 1 ) of isolate XC-6-3 and their parents had no significant differences. The results indicated that there may be diversity on the growth rate inheritance of MR mutants in asexual single-spore progeny. Table 3 Aggressiveness of MR mutants of Phytophthora boehmeriae to cotton seedling leaves WW-13-1, WW-13-3, XC-6-2, XC-6-3, WH-2-2, SX-9-2, and SX-9-4 are the obtained MR mutant strains. WW-13, XC-6, WH-2, and SX-9 are MS wild-type isolates of P. boehmeriae, and used as the parental control isolates. Data in the same column followed by different lowercase and capital letters are significantly different at α = 0.05 and α = 0.01 level by Duncan's LSR test  Table 4 Sporangium production of MR mutants of Phytophthora boehmeriae WW-13-1, XC-6-2, XC-6-3, WH-2-2, SX-9-2, and SX-9-4 are the obtained MR mutant strains. WW-13, XC-6, WH-2, and SX-9 are MS wild-type isolates of P. boehmeriae, and used as the parental control isolates. Data in the same column followed by different lowercase and capital letters are significantly different at α = 0.05 and α = 0.01 level by Duncan's LSR test  Table 5 Oospore production of MR mutant strains of Phytophthora boehmeriae XC-6-2, XC-6-3, WH-2-2, and SX-9-2 are the MR mutant strains. XC-6, WH-2, and SX-9 are MS wild-type isolates of P. boehmeriae, and used as the parental control isolates. Data in the same column followed by different lowercase and capital letters are significantly different at α = 0.05 and α = 0.01 level by Duncan's LSR test In the populations of the first single-oospore generation (OG 1 ) established with XC-6-2 and XC-6-3 single-zoospore generation (ZG 1 ) as the parents, and in the populations of the second single-oospore generation (OG 2 ) established with the first single-oospore generation (OG 1 ) of XC-6-3 as the parent, there were significant differences among the singleoospore isolates and their parents in the growth rates, and the variation in OG 2 (CV = 10.59%) was higher than that in OG 1 (CV = 5.68%).
Similarly, the growth rate of MS parent XC-6 of MR mutants displayed continuous variation in the single-zoospore progenies (ZG 1 , ZG 2 ) and single-oospore progenies (OG 1 ). These results indicated that the growth rate of MR and their parents displayed obvious variation or separation and were inherited unsteadily in their single-zoospore and single-oospore progenies.

Colony morphology
The colony morphology and its inheritance of MR mutants and their parents were compared in this experiment. The results showed that colony morphology of MR mutants could inherit steadily in the asexual single-spore progenies, but there were variations in single-oospore progenies. In addition to the parental type (type A: round colony, edges approximately neat, even-textured, with scant aerial mycelium), there were three variable types, type B, type C, and type D, in singleoospore progenies (Fig. 3). The colony of type B was irregular, rouge edges, uneven distribution of mycelia, with many aerial mycelia, and mycelium growing fast; the colony of type C was approximately round, irregular edge, with many aerial mycelia, and mycelium growing fast; and the colony of type D was approximately round, irregular edge, texture uniform, and radial mycelia obvious, with scant aerial mycelia (Fig. 3). In contrast, the colony morphology characters of wild-type parents, type A, were invariable in single-zoospore and single-oospore offspring. The results mentioned above suggested that as the resistance of P. boehmeriae to metalaxyl mutated, its colony morphology character also came to variation.

Homothallic character
Homothallic character and its inheritance of MR mutants of P. boehmeriae and their parents in their single-zoospore and single-oospore progenies were studied. Each isolate was Table 6 The inheritance of growth rate of MR mutants of Phytophthora boehmeriae in zoospore and oospore progenies ①ZG 1 , ZG 2 , OG 1 , and OG 2 stand for first and second single-zoospore progeny, first and second oospore progeny, respectively. ②** and * stand for significant level P < 0.01 and P < 0.05, respectively continuously determined for two generations, and 50 to 100 single-spore isolates were measured in single-zoospore and single-oospore progenies. All tested isolates produced oospores on LBA medium in single-isolate culture. The results showed that homothallic character of P. boehmeriae MR mutants could inherit steadily in the asexual and sexual single-spore progenies; in addition, the homothallic character of P. boehmeriae isolates was not changed with the mutation to metalaxyl.

Discussion
The effects of metalaxyl on genetic variation of the biological characters of oomycetes in Phytophthora were various and complicated. As mentioned above, in addition to resistance variation, the MR mutants in Phytophthora obtained from the field or laboratory mutagenesis could also display variations in other biological characters, such as aggressiveness/pathogenicity, sporulation capacity, spore germination, and culture characters. It was reported that the MR mutants of P. capsici grew much more slowly on metalaxyl-free V8 juice agar, and were less fit than the wild-type isolates (Bruin and Edington 1982). However, most studies showed that the MR isolates in Phytophthora were equal with and even stronger than the wild MS isolates in growth rate, sporulation, propagule germination, and pathogenicity to hosts (Bower and Coffey 1985;Gisi and Cohen 1996;Timmer et al. 1998;Café-Filho and Ristaino 2008;Qi et al. 2011;Wang et al. 2020b). Gisi and Cohen (1996) also found that colony morphology and growth rate of P. infestans produced variation in single-zoospore progenies. Especially, Wang et al. (2020bWang et al. ( , 2021 reported that the MR mutant strain SD1-9 of P. capsici was obtained by inducing on V 8 plate containing 10 µg·mL −1 metalaxyl from MS wild-type isolate SD1, which suggested that P. capsici was susceptible to high metalaxyl application induced metalaxyl resistance. In this paper, the MR mutants of P. boehmeriae were obtained by inducing on LBA amended with sublethal dose (10 µg·mL −1 ) of metalaxyl from the MS wild-type isolates successfully, which demonstrated that P. boehmeriae developed resistance to metalaxyl easily. This result was consistent with our results reported earlier (Gao et al. 1997;Chen et al. 2004aChen et al. , 2004b. It must be noted that we checked and determined if there was a mutation in the resistance inducing experiments based on the phenotypic character (metalaxyl resistance). The MS wild-type isolates were sensitive to metalaxyl (the minimal inhibited concentration of mycelial growth, MIC＜1 µg·mL −1 ), and did not grow on LBA containing 10 µg·mL −1 metalaxyl. The MR mutants of P. boehmeriae grew normally on LBA containing 10 µg·mL −1 metalaxyl. The method used by us is the classic one which is generally applied in the genetic study of fungicide resistance in Phytophthora. Indeed, there were no attempts to conduct any DNA sequence analysis of wild-type and MR isolates in this paper. However, the DNA sequence analysis will be possibly valuable, because it is possible that RPA190 gene also has a role in metalaxyl resistance in P. boehmeriae, as with P. infestans (Chen et al. 2018) and P. capsici (Wang et al. 2021). Therefore, characterizing the mutations in P. boehmeriae needs to be conducted in future.
Furthermore, the fitness and ability of the MR mutants of P. boehmeriae were studied in vitro. It was first reported the growth rate, zoospore production, temperature range for growth, the aggressiveness to cotton seedlings, and malachite green sensitivity of the MR mutants of P. boehmeriae had no significant difference compared with the MS wild-type parental isolates. Metalaxyl had no significant effects on the morphology and production of sporangia and oospores of MR mutants. However, the oospore production of MR mutants XC-6-2 and XC-6-3 was much lower than that of their parents, while the oospore production of MR mutants SX-9-2 has no significant differences with their parents. The results also showed that homothallic character of P. boehmeriae was steadily inherited in asexual and sexual progenies, and homothallic character did not change with metalaxyl-resistant induction. However, the oospore production of MR mutants was obviously inhibited by metalaxyl, and was various among the strains. The growth rates of MR mutants and their parents displayed obvious variation or separation and were inherited unsteadily in their single-zoospore and single-oospore progenies. The colony morphology character of MR mutants could inherit steadily in asexual single-zoospore progenies, but could not in sexual single-oospore progenies, while the colony morphology character of their parents could inherit steadily in asexual and sexual progenies, which showed that the inheritance of colony morphology was changed with metalaxyl-resistant mutation in P. boehmeriae.
Our research results suggested that the MR mutants of P. boehmeriae were as aggressive and fit as their MS wild-type parents in the absence of selection pressure from metalaxyl, which are consistent with the most related reports mentioned above. Since the MR mutant strains had a high level of resistance to metalaxyl, they would have much more competitive ability than the MS isolates in the selection pressure from metalaxyl in the field, and would probably develop the MR population easily in a short time. Thereby, there would be a high risk of development of metalaxyl resistance in populations of P. boehmeriae in the field, when metalaxyl was long and continuously applied for the control of the diseases caused by P. boehmeriae, which could lead to the defeat in controlling plant blight with metalaxyl. In addition, it must be noted that mefenoxam, metalaxyl-M, is the more recent isomer of metalaxyl, contains a much higher concentration of the active enantiomer of the original metalaxyl, and is therefore effective in the field (Parra and Ristaino 2001;Rubin et al. 2008;Hu and Li 2014;Lookabaugh et al. 2018;Gray et al. 2020;Gonzalez-Tobon et al. 2020;Siegenthaler and Hansen 2021).
The introduction of mefenoxam apparently led to a resurgence in use of both metalaxyl and mefenoxam in many regions in China. According to our recent survey, metalaxyl, mefenoxam, and metalaxyl (mefenoxam)-containing products were the most commonly used fungicides for control of cotton and ramie blight in the middle and lower reaches of Yangtze River, and some MR isolates of P. boehmeriae were detected in the field (unpublished data). Therefore, based on our research results and the present situation of metalaxyl (mefenoxam) resistance in P. boehmeriae, the monitoring and management of resistance in P. boehmeriae to metalaxyl (mefenoxam) should be strengthened urgently to avoid or lower the resistance risk and to ensure safety of cotton and ramie production. The measures for this included avoiding long-term and continuous use of metalaxyl, mefenoxam, and metalaxyl (mefenoxam)-containing products in the field, and practicing the alternation or combination of metalaxyl (mefenoxam) with other fungicides with a different action way for the control of the diseases caused by P. boehmeriae. Furthermore, the sensitivity of isolates of P. boehmeriae to metalaxyl (mefenoxam) in the field should be detected regularly to monitor the production and development of the resistant isolates timely and effectively.