Sexual Compatibility among Different Host-Originated Isolates of Aphelenchoides besseyi and the Inheritance of the Parasitism

Nine isolates of Aphelenchoides besseyi from two different hosts were studied. The isolates were identified at the species level according to morphometrics and fine structures observed under a scanning electron microscope. Two fern-originated isolates, Fu, and Fm, one rice-originated isolate, Rl, were not able to reproduce from a single juvenile, based on at least 50 replicates. The other six isolates were able to develop into a small population when inoculated with a single juvenile, demonstrating parthenogenesis. Crosses between isolates were conducted. In a compatibility cross experiment, three fern-originated isolates were selfed and crossed reciprocally, and all nine crossings had viable offspring. When fern isolates were used as paternal lines, the only two successful crosses were with the Rd line, and as maternal lines, only the Ff x Re and Fu x Rn crosses had viable offspring. Rl was used as the maternal line and Fm as the paternal line to study the inheritance of the bird’s-nest fern parasitism. Twenty of the 80 attempted crosses resulted in viable offspring and among these; six lines had the ability to parasitize on the bird’s-nest fern. When the F1 lines were back-crossed to the Rl maternal line, 20 viable offspring lines were obtained and among them 4 were able to parasitize bird’s-nest fern. These results indicate that bird’s-nest fern parasitism can be transferred to new generations by cross fertilization.

Variation in virulence and aggressiveness has been observed in many strains of both A. fragariae and A. ritzemabosi from different hosts and localities. This variation indicates great intraspecific heterogeneity, and might lead to variation in host range [16]. Noegel and Perry found that Aphelenchoides besseyi isolated from Chrysanthemum could not infect strawberry [17]; Rigges (1991) reported that A. besseyi isolated from strawberry could not infect rice, and vice versa [18]. These results show that A. besseyi, like Ditylenchus dipsaci, has different host preferences between isolates [19], but the existence of races of A. besseyi was not mentioned as a problem because the races were not differed by economically important hosts [17]. In Taiwan, A. besseyi was first reported on bird's-nest fern (Asplenium nidus L.) in 2002 and caused dark-brown patches on the leaves. This isolate was also capable of infecting strawberry and rice [3]. In the same study, a strawberry isolate was found to parasitize both rice and strawberry, but parasitism of the rice isolates was restricted to rice. Regardless of their ability to infect different hosts, these isolates did not differ in their morphology [3].
There have been no studies about the relationship between the Aphelenchoides besseyi host races and the mode of reproduction, or any attempts to cross isolates of A. besseyi to study the inheritance of the parasitism traits. The objectives of this study were (i) to conduct a cross fertilization experiment with A. besseyi isolates collected from different hosts and observe their compatibility, and (ii) to study the inheritance pattern of parasitism in A. besseyi by crossing rice and fern isolates.

Identification
The average morphometric measurements from the Fm isolate and the eight isolates obtained during 2004-2006, were within the range of measurements of the Aphelenchoides besseyi topotype described by Franklin and Siddiqi 1972 (data not shown). All nematodes had an off-set lip region, three to four pointed processes on the tail and four lateral incisures when observed under the cryo-field emission scanning electron microscope (data not shown). These characteristics were sufficient to support the species level identification [20].

Modes of Reproduction
Two fern-originated isolates, Fu and Fm, and one riceoriginated isolate, Rl, did not reproduce from a single juvenile in at least 50 replicates observed in this study. The other six isolates had replicates that were able to develop into a small population, demonstrating the ability to reproduce by parthenogenesis (Table 1). Among these parthenogenetic isolates, the Fk, Rp, Rn and Rd isolates had a lower percentage of replicates that were able to develop into a population from a single juvenile (Table 1).

Compatibility between Aphelenchoides besseyi Isolates
The fern-originated isolates were selfed and crossed reciprocally, and all nine combinations had viable offspring ( Table 2). When fern-originated isolates were used as the paternal line and five riceoriginated isolates as the maternal line, only the Rd x Fu and Rd x Fk crosses resulted in viable offspring. When fern-originated isolates were used as the maternal line and five rice-originated isolates were used as paternal lines, only the Ff x Re and Fu x Rn crosses had viable offspring (Table 3). When fern isolates were used as paternal lines, the only two successful crosses were with the Rd line yielding 33-83% viable offspring. The lowest rate of viable offspring was 17% when fern isolates were used as maternal lines ( Table 2).

Inheritance of Bird's-nest Fern Parasitism in Aphelenchoides besseyi
The first four batches of crosses between the Rl and Fm lines had 20 viable F 1 lines (Table 3), but batches four to eight did not result in any F 1 lines. When the 20 F 1 lines were used to inoculate a bird's-nest fern, lines 1-1, 1-2, 3-1, 4-1, 4-2 and 4-3 had the ability to parasitize fern (Table 3), causing typical symptoms to appear on the leaves (Fig 1A), in contrast with the F 1 lines that could not parasitize the fern, and did not show any symptoms ( Fig 1B).
When the six F 1 lines were back-crossed to the Rl maternal line, 120 attempted crosses resulted in 20 viable back-cross lines ( Table 4). The back-cross of F 1 1-16Rl had 8 viable offspring lines, and F 1 1-26Rl had 5 lines. The F 1 4-1, F 1 4-2 and F 1 4-3 backcrosses resulted in only one offspring line. The back-cross of F 1 3-16Rl did not result in any viable offspring. When the back-cross lines were inoculated on the bird's-nest fern, lines 11.7, 12.2, 31.1 and 42.1 were able to parasitize the fern (Table 4).  Table 2. The number of surviving and dying offspring lines from crosses of Aphelenchoides besseyi isolates.  Table 3. Successful crosses of Rl(R) and Fm(=) and their bird's-nest fern pathogenicity.

Batch number
The number of non-infectious F 1 lines The number of infectious F 1 lines (code)

Discussion
All nine isolates in this study were identified as A. besseyi based on morphometric data. However, the isolates varied in their abilities to infect the bird's-nest fern. The fern-originated isolates could parasitize strawberry and rice, but the rice-originated isolates could not parasitize fern [3,15]. Because nematode populations within plants are relatively isolated and often small, the chances are fairly high that favored genetic factors will increase and lead to intraspecific diversity. Almost all known cases of intraspecific variation in pathogenicity are confined to endoparasitic species [16]. A. besseyi is ectoparasitic for most of its life cycle in the majority of its hosts [7,12], and distinct food preferences between the isolates is a unique phenomenon.
In this study, most but not all isolates were able to reproduce from a single juvenile, indicating that some isolates might reproduce by cross fertilization, while the others have already evolved parthenogenesis. These results not only confirmed that the A. besseyi species has more than one mode of reproduction, but also showed two reproductive modes in one host race for the first time. Nematodes that belong to the same species but have two modes of reproduction are rare. Meloidogyne hapla has two biological races. Race A reproduces by facultative parthenogenesis and race B by obligate parthenogenesis [21,22]. The differences between these two races were found in the cytology of their ovaries. Facultative parthenogenesis involves meiotic division and obligate parthenogenesis uses mitotic division. In the studies of Meloidogyne genus, it was believed that species with sexual reproduction were more primitive than asexual ones [22]. Cayrol and Dalmasso (1975) studied the interspecific relationships among A. besseyi, A. ritzemabosi and A. fragariae and reported that crossing one juvenile from one of the species and ten males from another species gave positive results for five of the six possible crosses, and intermediate characteristics were observed in offspring [17]. Whether the species of the Aphelenchoides genus are of mixed origins like the Meloidogyne genus [23] and are in a transitional stage with both obligate cross fertilization and facultative (or obligate) parthenogenesis remains unanswered until further cytogenetic analysis of the nematodes can be performed.
In cross compatibility experiments, several combinations of crosses between fern-and rice-originated lines did not result in viable offspring; only four combinations produced a total of nine viable offspring lines (Table 2). On the other hand, 38 crosses were made from six combinations of the fern-originated isolates and resulted in 30 viable offspring lines, suggesting that the reproductive barrier between the nematode isolates from different hosts might form gradually. Organisms that move slowly may have developed into a new species after migrating to a new niche (parapatric speciation). Some parasites exhibit interspecific polymorphism, allowing individuals with a certain genotype to adapt to a new host (sympatric speciation) [24]. We observed that the Ff and Re isolates were able to reproduce vigorously by parthenogenesis (Table 1) but did not produce any viable offspring when their progenies were crossed ( Table 2). Similar results were observed in the Fk, Rn and Rp lines, indicating that parthenogenesis was inhibited by the presence of a male individual. The mechanism for this interesting phenomenon deserves further investigation.
The study of the inheritance of bird's-nest fern parasitism of A. besseyi is the first to be attempted in this genus. The parasitism behavior of above ground nematodes is quite different from that of underground, endoparasitic nematodes, such as Meloidogyne and Globodera spp. [25]. Unlike these obligate parasitic nematodes, the Aphelenchoides spp. are able to feed on fungal hyphae when host plants are not available, suggesting that the facultative parasitic Aphelenchoides spp. might use a very different strategy to cause plant diseases. Although a large number of crosses were made in this study, only a few viable F 1 offspring lines were obtained. Among them, we observed the cross between bird's-nest fern noninfectious Rl and bird's-nest fern infectious Fm resulted in viable offspring that were also able to parasitize bird's-nest fern. When these bird's-nest fern infectious F 1 lines were backcrossed to the Rl maternal line, four viable offspring lines inherited the ability to infect bird's-nest fern. These results indicate that bird's-nest fern parasitism can be transferred to new generations by cross fertilization.

Collection of Aphelenchoides besseyi Isolates
Eight isolates of Aphelenchoides besseyi from two different hosts were studied during 2004-2006 (Table 5). The Fm isolate was obtained 2009, and therefore not included in the compatibility test. A. besseyi isolates were collected from the infected rice grain or fern leaves. Diseased tissue was washed several times with distilled water to clean the surface, immersed in distilled water, and cut coarsely to release the nematodes. Nematodes were observed after 5-10 min and transferred onto PDA slants containing Alternaria citri. The nematode cultures were maintained at 24uC without light to increase the population. The isolates were identified at the species level. Nematodes were sterilized with 1000 ppm malachite green and 2000 ppm streptomycin, and maintained on Alternaria citri PDA slants [3] in a 24uC growth chamber.

Analysis of Reproduction Modes of Aphelenchoides besseyi Isolates
Before the crossing experiments could be performed, the mode of reproduction of the A. besseyi isolates needed to be clarified. Single juveniles were transferred onto Alternaria citri PDA slants, with at least 20 replicates for each isolate. The cultures were incubated without lights at 24uC for 14 to 28 days before examination. If a single juvenile was able to reproduce, multiple nematodes would be present in the PDA slant tube, indicating that an isolate was capable of reproducing parthenogenetically. More replicates were performed if the isolates did not reproduce in the first 20 replicates. A maximum of 100 replicates were performed.

Compatibility between Aphelenchoides besseyi Isolates Originated from Different Hosts
Three bird's-nest fern isolates were selfed and crossed reciprocally for nine combination crosses. The three bird's-nest fern isolates, Ff, Fu and Fk, and five isolates from rice were used as parental lines. A total of 30 combination crosses were conducted, and all crosses had six to eight replicates. One juvenile from the maternal line and one adult male from the paternal line were transferred onto a Alternaria citri PDA slant to conduct the cross fertilization. The crosses were maintained under the same conditions described in the previous paragraph and observed after 14-28 days. All of the cultures were observed under a dissecting microscope. If no nematodes were observed, some distilled water was added to the tube and vortexed several times to wash the culturing slant for further observation.

Inheritance of the Bird's-nest Fern Parasitism in Aphelenchoides besseyi
Based on the results from the reproduction mode experiments and the previous host range studies, the Rl isolate from rice and the Fm isolates from bird's-nest fern were chosen for the cross. Rl which could not infect bird's-nest fern was used as the maternal line, and Fm, which could infect bird's-nest fern, was used as the paternal line to ensure that the offspring that obtained the parasitism trait was from a successful cross. Eight batches of crosses were conducted at different times and 10 crosses were made in each batch. The viable offspring were maintained on Alternaria citri PDA slants to increase nematode population, and challenged on bird's-nest fern by injecting approximately 1000 nematodes into the leaf [3]. The F 1 lines that were able to infect bird's-nest fern were used as paternal lines and Rl was used as the maternal line in the back-crosses. For each combination, 20 crosses were made. All viable backcross lines were maintained on Alternaria citri PDA slants to increase nematode population and challenged on the bird's-nest fern to determine their parasitic ability.