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Journal of Nematology 40(3):226239. 2008. The Society of Nematologists 2008.

Distribution, Biology and Pathology of Anguina pacicae


MICHAEL A. MCCLURE,1 MARK E. SCHMITT,1 MICHAEL D. MCCULLOUGH2
Abstract: Anguina pacicae is distributed along a narrow strip on the Pacic coast of Northern California where it forms galls on the shoots of Poa annua and causes signicant damage to golf course greens. Methods were developed for the continuous propagation of A. pacicae on P. annua in growth chambers, and they were used to examine the life cycle and host-parasite relationships of the nematode. At a mean temperature of 208C (228C day/188C night) the life cycle was completed in as little as 32 days (inoculation to second-generation J2). The rst molt occurred in the egg. Infective J2 hatched from the eggs and penetrated the shoot near the crown of the plant where a cavity was formed 200 to 300 mm below the shoot apex. A gall around the cavity was visible 12 days after inoculation (DAI), and the cavity and gall continued to enlarge until second-generation J2 began to hatch. Three additional molts occurred in the cavity of the developing gall 14 to 24 DAI. Sexes could be distinguished 15 DAI. Egg production began 26 DAI and continued for 10 to 15 days. Eggs commenced hatching inside the gall 42 DAI, when the adults began to die and decompose. By 57 DAI, the gall had reached its maximum diameter, and the cavity was lled entirely with second-generation J2 that remained in the gall until they were liberated when the gall decomposed. J2 in galls survived desiccation over silica gel for 14 months at 148C and were active and infective when rehydrated. Key words: Agrostis, Anguina pacicae, anhydrobiosis, annual bluegrass, distribution, etiology, host-parasite relationship, host range, life cycle, Lolium perenne, Pacic shoot-gall nematode, Poa annua, Poa trivialis.

Anguina pacicae currently is the most devastating pest of Poa annua (Poa) putting greens on Northern California golf courses. On Poa, it induces stem galls at the crown of the plant that sequentially contain developing juveniles, adults, eggs and second-generation J2. Initial symptoms on turf consist of small yellow patches, 25 to 75mm in diameter, which enlarge and may coalesce as the nematodes spread (Fig. 1). Young, infected plants may die and, when the infestation is severe, a rough, uneven putting surface results. First discovered in 1978, the nematode was initially identied as Anguina radicicola (Costello, 1983). Subsequently, it was characterized morphologically and described as a new species (Cid del Prado and Maggenti, 1984). A survey, conducted in 2005, found 12 infested golf courses, all in the coastal California counties of Monterey, San Francisco and San Mateo (Westerdahl et al., 2005). Early investigations of A. pacicae, most of them unpublished or published in trade magazines and newsletters, were concerned with its distribution (Costello, 1983; Westerdahl et al., 2005), population dynamics (E. Giat, R. Kaspi, C. A. Anderson and B. B. Westerdahl, pers. comm.), and methods of control (Peterson et al., 1986; Stowell, 1998; Westerdahl et al., 2005). Based on eld observations and knowledge of related species, Stowell (1998) proposed a potential life cycle, but details were not determined. In this study, we reviewed recent records of infestations and investigated the nematodes host range, life cycle, biology and histopathology on P. annua.

MATERIALS

AND

METHODS

Received for publication November 25, 2008. 1 Department of Plant Sciences, University of Arizona, Tucson, AZ 85721. 2 Northern California Golf Association, P.O. Box NCGA, Pebble Beach, CA 93953. Funding for this research was administered by the Northern California Golf Association with contributions from industry, individual golf courses and professional associations. The authors thank Mark M. Mahady and Larry J. Stowell for their advice and assistance. E-mail: mcclure@ag.arizona.edu This paper was edited by David Bird.

Distribution: A distribution map was compiled from published records, positive diagnoses on samples submitted to our laboratory, and records provided by the Northern California Golf Association (Pebble Beach, CA), and the PACE Turfgrass Research Institute (San Diego, CA). Stock cultures: Plugs of Poa infected with A. pacicae were obtained from the Number 12 green at the Cypress Point Golf Club, Pebble Beach, CA. Infective J2 were dissected from mature galls and used to inoculate Poa seedlings. Stock cultures of infected plants were maintained in 1.0 ml plastic pipette tips (Fisher Scientic, Pittsburgh, PA) containing 2 g of a 3:1 mixture of 30-mesh and 60-mesh quartz sand. Fifty tubes were placed in the rack of the original pipette storage box to which 350 ml of nutrient solution [1 mM Ca(NO3)2, 1 mM K(NO3), 0.5 mM KH2PO4, 0.25 mM MgSO4, 0.1 mM K2SiO3, 10 mM Fe(NO3)3, 25 mM Fe-HEDTA, 3 mM MnCl2, 4 mM ZnSO4, 2 mM H3BO3, 1 mM CuSO4, 0.09 mM Na2MoO4, pH 5.6] was added so that the bottom 7 mm of the tubes were submerged and the sand was wetted to the top by capillarity (Fig. 2). One or two Poa seeds, cv. True Putt, (DLF International Seed, Halsey, OR) were placed in each tube and covered with an additional 0.15 g of the sand mixture. The planted boxes were kept in a growth chamber with a day temperature of 228C, a night temperature of 188C (12-hour cycle) and an average relative humidity of 75%. Daytime light intensity in the growth chamber was 215 mMol/m2/sec. With the lid of the pipette box closed, light intensity inside the box was 175 mMol/m2/sec. Evaporation of the nutrient solution was minimized by closing the lid, and it was not necessary to replenish the solution for up to 10 wk. The high humidity inside the box also favored infection by A. pacicae. When the seedlings were 14 days old, the nutrient solution in the pipette box was discarded, and the seedlings inoculated by pipetting 25 to 30 J2 onto the sand at the crown of

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Biology of Anguina pacicae : McClure et al. 227 the plant. Four days after inoculation (DAI), the nutrient solution was replaced. Unless the nutrient solution was removed during the rst 4 d, most of the J2 migrated from the sand into the nutrient solution. Infective, second-generation J2 were harvested from mature galls 60 to 80 DAI. Host Range: Twenty-eight cultivars and accessions of bentgrass, Agrostis spp., were assayed for susceptibility to A. pacicae. They included Tyee Creeping Bent, Brighton, Alpha, SR 11502, Dominant Extreme, Legendary, MacKenzie Creeping Bent, SR 1119 Creeping Bent, SR 7200 Velvet Bent, SRX 70D Dryland Bent, Red Top Bent, Sandhill Creeping Bent, SR 7150 Colonial Bent, 007 Creeping Bent, Penn A-4, Penn A-1, Penn A-2, Penn G -1, Penn G -2, Penn Eagle, Penn Links II, Independence, Bengal, Dominant Spring, Seaside II, Bar King, T1 and Shark. Perennial rye grass, Lolium perenne, cv. Barlady and cv. Bargold and Poa trivialis cv. Sabre were also tested. All tests were conducted in the pipettetip containers maintained in a growth chamber under the conditions described above for maintenance of stock cultures. Life cycle: Poa seedlings in pipette-tip containers were inoculated 14 d after planting and placed in three separate growth chambers, each programmed with a different temperature regime: 228C day/188C night, 188C day/148C night and 148C day/108C night with a 12 hr day/night cycle at a daytime light intensity of 215 mMol/m2/sec and 75% relative humidity. Every other day, ve developing galls were dissected in water and the nematodes hand-picked into xative containing 4% formalin and 1% glutaraldehyde in 0.01 M phosphate buffer, pH 7.3. Fixed specimens were stored in xative at 48C for up to 4 mon until they could be photographed and measured. Growth rate was determined by measuring the outline body area of the nematodes. Ten of the largest nematodes from each harvest date were

FIG. 1. Anguina pacicae on Poa annua. (A) Damaged putting green. (B) Galls on the crowns of infected plants (arrows).

FIG. 2. Pipette box assembly for rearing Anguina pacicae. (A) Pipette tip container (Clear, translucent lid removed for photo). (B) One-ml pipette tips containing quartz sand and a Poa annua seedling.

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FIG. 3. Distribution of Anguina pacicae in California (shaded area).

mounted on glass microscope slides, and the coverslips supported with small pieces of stainless steel wire or glass rods drawn to a diameter slightly larger than that of the nematodes. When the sexes could be distinguished, 14 DAI and later, males and females were mounted on separate slides. Mounted nematodes were viewed and photographed on a compound microscope tted with an Olympus DP71 digital camera (Olympus America, Center Valley, PA). Nematode body area was calculated from the digital images with LabWorks image analysis software (UVP Inc., Upland, CA). Because nematode development in any given gall was not synchronized, it was difcult to determine by direct observation when the 3rd and 4th molts occurred, so the timing of the molts was estimated by conducting a separate experiment at 22/188C and dissecting 10 galls each day from 10 to 20 DAI. All the extracted nematodes were xed and mounted on slides, as described above, and examined under the compound microscope at magnications of X160 to X400. Each day,100 or more nematodes were examined. All molting juveniles were photographed, and the images were used to measure their body areas digitally. Evidence of molting included the presence of a partially cast cuticle or the absence of a fully formed stylet. Female fecundity: Egg production at 22/188C was measured over a 10-d period. Each of 10 plants was inoculated with approximately 30 J2. Galls were harvested beginning 20 DAI and at 2 or 3-d intervals thereafter for another 18 d. Individual galls were dissected in water, and the numbers of males, females and eggs counted. The experiment was terminated 38 DAI when females

FIG. 4. Growth of Anguina pacicae at three temperature regimes. (A) 228C day/188C night. (B) 188C day/148C night. (C) 148C day/ 108C night. Dotted arrows = rst appearance of eggs. Solid arrows = rst appearance of second-generation infective juveniles. DAI = days after inoculation.

began to decompose and it became difcult to distinguish them from decomposed males. Infectivity of J2: During the course of these experiments, it was noted that many of the J2 dissected from galls between 40 and 45 DAI were less active and more

Biology of Anguina pacicae : McClure et al. 229

FIG. 5. (continued on next page).

230 Journal of Nematology, Volume 40, No. 3, September 2008

FIG. 5. Reproductive structures in Anguina pacicae. A-H and K-N, 22/188C (day/night). I-J, 18/148C (day/night). (A) Male during 4th molt. (B) Female at the beginning of 4th molt. (C) Adult male with sperm (arrows) in vas deferens. (D) Adult female with sperm-like bodies (sb) in post-uterine sac. (E) Adult female with sperm (arrows) in uterus and post-uterine sac. (F) Adult male vas deferens packed with sperm. (G) Adult female with single-cell eggs in uterus. (H) Adult female with sperm (arrows) in spermatheca (st) at its junction with the ovary (ov). I) Postuterine sac containing globular refractive bodies (sb). (J) Adult female with sperm (arrows) in uterus and post-uterine sac. (K) Adult female beginning to decompose. (L) Adult female cuticle containing second-generation J2. (M) Newly laid eggs with 1, 2 and 3 cells. (N) Adult female with two-cell eggs in uterus.

translucent than older J2 dissected from galls 50 to 60 DAI. Their appearance was similar to that of J2 that had emerged from eggs dissected from galls and hatched in distilled water. To determine if these J2 were infective,

several thousand eggs were collected from galls 36 DAI and hatched on a nylon mesh sieve (20 mm-pore-size) suspended on the surface of demineralized water. The hatched J2 were collected and used to inoculate Poa

Biology of Anguina pacicae : McClure et al. 231 the remaining four rehydrated after 17 mon. Dried galls, stored on the laboratory bench, were rehydrated after 17 mon. Rehydrated, active J2 were used to inoculate 14-d-old Poa seedlings. Similar procedures were used to test the ability of free, active J2 to withstand desiccation. Fresh galls were dissected in water, and the liberated J2 were rinsed and concentrated by centrifugation. Approximately 300 J2 in 50 ml of distilled water were pipetted onto the center of plastic coverslips, 25-mm-square, where the water maintained a cohesive bead covering the nematodes. The coverslips were placed in sealed glass jars at 70% RH and incubated at 158C. After 48 hr, all of the water had evaporated, and there was no visible surface moisture on the coverslip. At intervals, the nematodes on the coverslips were rehydrated by covering them with 100 ml of water and incubating them at 228C for 4 hr. The number of J2 that revived was counted, and then 50 J2 from each coverslip were used to inoculate Poa seedlings maintained at 22/188C. Inoculated seedlings were dissected 30 DAI to determine the infectivity of the revived J2. Histopathology: Poa seedlings in pipette-tip containers were inoculated, as described above. Every other day, following inoculation, 10 plants were harvested and rinsed under a stream of distilled water to remove sand adhering to the roots. Shoots and roots were excised 2 to 3 mm above and below the crown and discarded. The infected crowns, with the remnants of roots and shoot attached, were xed in FPP (formalin:isopropanol: proprionic acid), dehydrated in isopropanol and embedded in Paraplast X-TRA (Daykin and Hussey, 1985). A minimum of 5 crowns from each harvest were sectioned. Serial sections of embedded crowns were cut 10-mm thick on a rotary microtome, and ribbons of sections were oated on a pool of 0.03% gelatin in demineralized water spread on glass microscope slides. The slides were dried at 388C overnight, and the sections were stained with Triarch Quadruple Stain (Daykin and Hussey, 1985). Coverslips were mounted with Permount (Fisher Scientic, Fair Lawn, NJ). Additional sections were stained with aniline blue to detect callose by visible light and UV uorescence microscopy (Jensen, 1962). Whole mounts of infected crowns were prepared by xing the crowns for 7 or more d in FPP, staining them in 0.01% aqueous aniline blue (Aldrich Chemical Company, Milwaukee, WI) for 48 hr, destaining them in lactophenol until the plant tissues were clear, and mounting them on glass slides in lactophenol. RESULTS Distribution: Anguina pacicae was found on 31 golf courses in Northern California, from Mendocino in the north to Carmel in the south (Fig. 3). Its range was limited to a narrow strip along the coast within a few kilometers of the Pacic Ocean. The most inland site was approximately 30 km from the coast at Santa Rosa.

FIG. 6. First molt in eggs of Anguina pacicae. (A) Molting rststage juvenile with cast cuticle (arrow) around the head and an incompletely formed stylet (s). (B) Second stage juvenile with fully formed stylet (s).

seedlings. J2 from 60-d-old galls were used for controls. Sixty DAI the plants were dissected to determine the state of nematode development. Anhydrobiosis: Galls from stock cultures, 60 DAI, were slowly dried by reducing the ambient relative humidity (RH) from 90% to 10% over a 20-d period. Relative humidity was controlled by placing the galls on lter paper suspended over sodium hydroxide solutions in sealed glass jars, with the concentration of NaOH adjusted to achieve atmospheric moisture levels of 90%, 70%, 50%, 30%, and 10% RH at 158C (Madge, 1961). Galls were held for 4 d at each level of RH and an additional 10 d at 10% RH. After 14 days at 10% RH, a few of the galls were rehydrated overnight on moist lter paper at 48C, and the J2 were liberated by dissection in distilled water. Eight dried galls were transferred to sealed jars containing silica gel desiccant and stored at 148C. Another four were stored on the laboratory shelf without temperature or humidity control. Four galls dried over silica gel were rehydrated after 11 mon, and

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FIG. 7. Post-infection molts of Anguina pacicae. (A) Infective J2, 2 days DAI. (B) Newly formed J3 bearing cast cuticle of J2. (C) Newly formed J4 bearing cast cuticle of J3. (D) Pre-adult female bearing cast cuticle of J4. The vulva, vagina and developing gonad are visible. Note that the nematode in 7D was photographed at a lower magnication than those in 7A-C. Printed to scale, the nematode would be 2.5X larger than shown.

All of the infections were on Poa greens and their surrounding collars. None were found on bentgrass (Agrostis stolonifera) or other grasses in or around the putting greens. To date, this nematode has not been found in Oregon (S. C. Alderman, personal communication). Host Range: From a total of 10 replicate plants, only one plant of each of the following bentgrass cultivars had a gall containing eggs: Tyee, Brighton, Dominant Extreme, Shark, Alpha, Legendary, and SR 11502. The remaining cultivars had none. Poa trivialis was also highly resistant. Only three plants out of 48 had galls containing eggs or second-generation J2. No galls were found on Lolium perenne. Eighty to ninety percent of Poa seedlings used as controls in these tests were infected, and the nematodes in galls completed their life cycle. Life Cycle: Growth and development of A. pacicae was dependent upon temperature. With day-night temperatures of 22/188C, little increase in body area was observed for the rst 6 to 8 DAI (Fig. 4). After 10 days, growth was rapid and, 24 DAI, both sexes had reached maximum body size. At maturity, the body area of fe-

males was up to three times that of males. Sexes could be distinguished 14 to 16 DAI, when the gonads and genitalia had begun to develop (Fig. 5A,B). Sperm were observed in the vas deferens of the male and sperm-like bodies were detected in the post-uterine sac of the female 20 DAI (Fig. 5C,D). The vas deferens became packed with sperm, and sperm were also found along the entire length of the female oviduct, including, the spermatheca (Fig. 5E-J). Eggs rst appeared in the gall 20 DAI, and 29 DAI there were hundreds of eggs in each gall. Growth was essentially complete 26 DAI, but females continued to lay eggs until 36 DAI, after which they began to senesce (Fig. 5K) and body area decreased slightly (Fig. 4). By 44 DAI, most of the adults, both male and female, were moribund and partially decomposed. Second-generation J2 were observed 32 DAI, and 44 DAI galls contained mostly J2, along with a few eggs. In older cultures, the exoskeletons of a few of the moribund females contained living J2 (Fig. 5L). The appearance of the post-uterine sac contents changed during maturation and aging of the female.

Biology of Anguina pacicae : McClure et al. 233


TABLE 1. Molting of Anguina pacicae in Poa annua at 22/188C (day/night).
Mean body area of 10 largest nematodesb,c,d

TABLE 2.

Fecundity of Anguina pacicae females on Poa annua.


Mean number of males per gall Mean number of females per gall Mean number of eggs per gall Mean number of eggs per female

DAI

Number of nematodes observed

Number of nematodes molting

Percentage of nematodes molting

DAIa

Number of galls with eggs

10 11 12 13 14 15 16 17 18 19 20
a b

201 240 179 123 182 247 248 210 126 184 150

9 82 55 46 42 95 69 65 39 36 7

4 34 31 37 23 38 28 31 31 20 5

18 20 23 37 47 111 97 103 120 102 109

20 22 24 27 29 31 34 36 38
a

2 6 9 9 9 8 9 9 9

11 5 5 3 4 4 3 3 4

11 4 4 4 4 4 3 3 4

110 220 524 1107 1712 1956 1746 2409 2568

10 55 131 369 428 489 582 803 642

DAI = days after inoculation. Ten galls examined at each harvest.

DAI = days after inoculation. Ten galls dissected at each harvest interval. Body area =mm2 X 1000 c Less than 10 measured: 10 DAI = 9 nematodes; 16 DAI = 7 females; 20 DAI = 4 females. d Males and female juveniles could not be distinguished up to 14 DAI. After 14 days, only females were measured.

Soon after the last molt, sperm-like bodies were observed in the post-uterine sac and uterus (Fig. 5D,E). Later, although these sperm-like bodies could still be found (Fig. 5J), the post-uterine sac of many females contained globular, irregularly shaped bodies that did not resemble sperm (Fig. 5I). The morphology of the sperm changed following insemination of the female. Sperm in the male were roughly spherical (Fig. 5F), becoming oblong as they passed through the constricting lumen of the vas deferens (Fig. 5C). Spherical sperm were also found in the uterus of the female (Fig. 5J), but sperm that had migrated to the spermatheca were compressed and angular (Fig. 5H). The overall developmental pattern was similar at lower temperatures but with a corresponding delay in all stages of growth (Fig. 4B,C). At day/night temperatures of 18/ 148C, the initial growth lag was protracted to 14 d and sexes could not be distinguished until 18 DAI. Eggs rst appeared 24 DAI and second-generation J2, 36 DAI. At day-night temperatures of 14/108C, the initial growth lag was extended to 16 DAI, eggs rst appeared 26 DAI, and second generation J2, 46 DAI (Fig. 4C). Adults from plants reared at 14/108C were larger than those reared at 18/148C or 22/188C. Calculated from the time of inoculation to the rst appearance of second generation J2, the life cycle took 32 d at 22 to 188C, 36 d at 18 to 148C and 46 d at 14 to 108C. Molting: The rst molt occurred in the egg (Fig. 6), and subsequent molts were completed after the hatched J2 had infected the plant and formed a cavity within the shoot. At 22/188C, the second molt was rst observed 10 DAI, the third molt 13 to 14 DAI, and the fourth molt 15 to 19 DAI (Fig. 7). Molting intervals could be estimated by measuring the body area of developing nematodes from 10 to 20 DAI (Table 1). Molting J2 had an average body area of 20,000 mm2,

molting J3 an average body area of 42,000 mm2, and molting J4 an average body area of 108,000 mm2. Molting juveniles were found in galls more than 20 DAI, but their small size and juvenile anatomy indicated that they were individuals whose development had been retarded compared to the largest nematodes in the gall. Female fecundity: Oviposition began 20 DAI and increased rapidly over the next 10 d at 22 to 188C (Table 2). Although eggs continued to be laid, the experiment was terminated 38 DAI because some adults had begun to decompose (Fig. 5K,L), and it was difcult distinguish the sexes when only twisted cuticles remained. When plants were inoculated with 30 J2, galls contained an average of 4.6 adult females and, by 38 DAI, 2,568 eggs. Females laid up to 1,000 eggs, with some individuals producing as many as 1,200. Females dissected from galls and mounted in water on microscope slides were observed to lay up to 4 eggs/hr which, by extrapolation, would yield 1,000 eggs over 10 d. A 1:1 sex ratio was observed in this experiment and conrmed by general observation throughout the course of the investigation. Occasionally, galls were dissected that contained a single male or single female, 30 or more DAI. Less frequently, galls contained two females. Single-nematode galls or single-sex galls never contained eggs, and females in single-female galls never contained developing eggs in their uteri. Eggs dissected from galls and hatched in vitro produced J2 that were similar in appearance to J2 dissected from galls soon after they had hatched within the gall. Both were pale and devoid of the intestinal granules that characterize infective J2 (Fig. 8). Several hundred eggs were hatched in vitro, and the J2 maintained in distilled water at 148C for 10 d. Their appearance did not change, and they gradually became less active. All 10 of the Poa seedlings inoculated with J2 collected from mature galls (control inoculum) were galled and contained hundreds of second-generation J2, whereas J2 hatched in vitro did not induce galls, and no nematodes were found in these plants 60 DAI. Anhydrobiosis: Rehydrated J2, recovered from galls that were dried for 2 wk at 10% RH, infected nine out of

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FIG. 8. Second-stage juveniles of Anguina pacicae. (A) Hatched naturally in a mature gall, 60 DAI. (B) Hatched from eggs extracted from galls and incubated in distilled water. Note refractive granules in the intestine of J2 hatched in vivo and hyaline appearance of J2 hatched in vitro.

10 Poa seedlings and completed a normal life cycle. Rehydrated J2 from galls dried an additional 2 wk over anhydrous silica gel infected ve out of seven Poa seedlings. After 11 mon over silica gel, 60% of the J2 were active and infected Poa when released from rehydrated galls. But, after 17 mon, fewer than 1% were active and none infected Poa. Infective J2, liberated from fresh, mature galls and dried on plastic coverslips at 70% RH for 2 to 8 d, regained activity within 2 hr when rehydrated and infected Poa seedlings. After 16 d at 70% RH, only 16% of the J2 became active when rehydrated and their movements were slow and barely perceptible. None infected Poa seedlings. Histopathology: Within 48 hr after inoculation at 22 to 188C, J2 entered the shoots of Poa seedlings by passing between the leaf sheaths (Fig. 9A), where they were found 1 to 2 mm above the plants crown, migrating downward (Fig. 9B). Four DAI, they had penetrated tissues of the crown, separating cells along their path of migration, and had reached a position 100 to 200 mm below the shoot apex. A cavity surrounding the nematodes was initiated beneath the apex by dissolution of the middle lamellae and collapse of punctured cells. Six DAI, the cavity had reached a volume of 0.002 to 0.003 mm3 and contained up to 12 developing juveniles (Fig. 9C). By 12 DAI, the J2 had increased in size, and swelling (galling), resulting from hypertrophy and hyperplasia of the cortical cells of the shoot above the crown, was apparent (Fig. 9D). The cavity continued to enlarge as the nematodes matured, and the rst eggs were found in the cavity 20 DAI. Twenty-nine DAI, all of

the nematodes were adults, and the gall had swollen to more than 2.0 mm in diameter with the cavity occupying more than half of the galls overall volume (Fig. 9E). In almost every case, the cavity contained an air bubble, 0.1 to 0.2 mm in diameter. Thirty-six DAI, the cavity was lled with eggs that began to hatch 43 DAI (Fig. 9F). Second-generation J2 that hatched from the eggs initially were found primarily near the periphery of the cavity (Fig. 9F,G) but, by 57 DAI, the entire cavity was lled with uncoiled J2, many arranged in parallel arrays (Fig. 9H). At this point, only remnants of the adults were found. A layer of cells, 3 to 5 cells thick, surrounded the cavity 50 DAI. The cells were lled with cytoplasm and stained more intensely than cells further from the cavity (Fig. 9G). Dark-staining, amorphous material lined the cavity discontinuously as the J2 began to hatch (Fig. 9F,I) and it persisted until the J2 had matured. This material did not stain with aniline blue and did not uoresce in sections incubated in aniline blue. The cavity frequently extended to just beneath the epidermis of the gall (Fig. 9J), but the J2 did not exit the gall until the plant tissues began to decompose. After 80 d, J2 began to emerge from the decomposing gall, and, occasionally, they induced a secondary gall 0.25 to 0.5 mm higher on the shoot. DISCUSSION Golf is a major industry in California, where it supports 160,000 jobs and contributes more than $6 billion

Biology of Anguina pacicae : McClure et al. 235

FIG. 9. (Continued on next page).

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FIG. 9. (Continued on next page).

Biology of Anguina pacicae : McClure et al. 237

FIG. 9. Histopathology of Anguina pacicae on Poa annua. (A) Infective J2 passing between leaves of the shoot, 2 mm above the crown, 2 DAI. B-H, longitudinal sections through the crown of the plant. (B) Two DAI, J2 (arrows) are migrating downward between the leaf sheaths. (C) Six DAI, J2 have initiated a cavity (c) just below the shoot meristem. (D) Hyperplasia of shoot parenchyma cells, 12 DAI, results in visible swelling at the base of the stem. Cavity volume increases. (E) Adults in the cavity at the onset of egg laying, 29 DAI. Cavity contains loose parenchyma cells (arrows) and a few eggs. (F) At 43 DAI, the cavity is lined with a discontinuous, dark-staining, amorphous layer (see also Fig. 9I) and is becoming lled with eggs. (G) Eggs begin to hatch, 50 DAI, and a layer of cells, lled with cytoplasm and 3 to 6-cells thick, surrounds the cavity (see also Fig. 9I). (H) Cavity is lled with second-generation J2 laying uncoiled and often in parallel arrays, 57 DAI. (I) Detail of cells surrounding cavity, 50 DAI, showing the dark-staining amorphous layer (arrow) and large parenchyma cells containing dense cytoplasm and prominent nuclei. (J). Cavity containing J2 extends to the outer wall of the gall, 57 DAI. The epidermis (ep) of the gall is intact.

annually, directly to the economy. In Northern California alone, there are an estimated 1.3 million golfers who play on 399 courses on which Poa accounts for more than 75% of the turf grass maintained on putting greens. A. pacicae is a major threat to those greens in coastal areas. Our discovery of infestations as far north as Mendocino showed that its distribution is much greater than originally determined and its ability to reproduce at an average temperature of 128C increases the likelihood that infestations will eventually be found in the states of Oregon and perhaps Washington. Poa trivialis Sabre and certain Agrostis cultivars proved to be poor hosts for A. pacicae. However, the nematode was able to complete its life cycle in a few individual plants of several of the Agrostis cultivars. This suggests that the seed lots we used were genetically variable with regards to susceptibility. An alternative hypothesis is that individual nematodes possess complementary genes for virulence. If this is the case, it can be predicted that repeated exposure of these grasses, nominally resistant to A. pacicae, will result in populations of the nematode with elevated frequencies of individuals pathogenic to those particular cultivars. Golf course superintendents who consider replacing infested Poa greens with bentgrass turf must consider this possibility. Nematode development was measured by plotting outline body area over time. Development of individual nematodes inside a given gall was not synchronized. Galls containing adult females 18 DAI also contained juvenile females and a few sexually undifferentiated juveniles. The occurrence of several growth stages inside a

gall resulted, in part, from J2 reaching their feeding site over a 2-to 3-day period. At 22/188C, serial sections through galls, 4 to 6 DAI, occasionally showed J2 still migrating downward between leaf sheaths and through tissues of the crown, while others had already reached their feeding site beneath the shoot apex. This, and competition between nematodes for nutrients and space within the gall, would have contributed to growth differences at any given sampling date. It has been reported that the J1 emerges from the eggs of A. tritici and that the rst molt occurs after hatching ( Jones and Jones, 1974; Hooper and Southey, 1978; Agrios, 1997). But careful examination of developing A. tritici eggs clearly demonstrated that the rst molt occurs in the egg (McClure, 1988), a developmental sequence also documented for A. agrostis (Stynes and Bird, 1982) and for A. pacicae, during our investigation. Based on the failure of females to reproduce when only a single female occupied a gall and on several failed attempts to infect Poa with a single, infective J2 (data not shown), we concluded that reproduction is amphimictic. In the absence of males, females never produced eggs, nor did they contain developing eggs in their uteri, suggesting that fertilization is required for oocytes to pass from the ovary to the spermatheca. When males were present, sperm were easily observed in the uterus and oviduct of the female, but more than 30 or 40 in the females entire gonad were uncommon. Because females lay up to 1,000 eggs over a 10-day period, we concluded that multiple matings must occur and, considering the proximity of males to females,

238 Journal of Nematology, Volume 40, No. 3, September 2008 multiple mates also are possible, if not likely. An alternative hypothesis is that sperm are simply deposited within the cavity of the gall and make their own way to the vulvae of the females. However, serial sections cut through galls during the reproductive period did not reveal any such sperm. Structures with the appearance of sperm were found in the females post-uterine sac, signifying that this structure can serve as a seminal receptacle. Irregular, globular bodies, also observed in the post-uterine sac, have been described as degenerate cells (Cid del Prado and Maggenti,1984). During oviposition, a small jet of semi-viscous material always preceded extrusion of an egg from the vulva. While it is tempting to speculate that this material may have originated from the globular bodies in the post-uterine sac, there is no evidence for such a conclusion, and the origin of the material remains unknown. Second-generation J2 extracted from mature galls readily infected Poa, whereas J2 hatched in vitro from eggs did not. The translucent appearance of the latter suggested that their development was not complete. Most notable was the complete lack of refractive granules in their intestines. Because the appearance of these J2 did not change when they were stored in demineralized water, we concluded that J2 hatched in the gall must obtain some essential nutriment either from the plant tissues or from the decayed adults. At 22/188C this occurred during the 24 days following the appearance of the rst J2 (32 DAI) and the cessation of hatching (57 DAI). A developing gall formed by Anguina tritici in wheat orets also contains a cavity. It is lined by granular cells that form a nutritive layer on which nematodes feed ( Jones and Jones, 1974). A layer of material that lined the cavity formed by A. agrostis on annual ryegrass was identied as callose, which was thought to form as plant response to wounding and cellular damage (Stynes and Bird, 1982). A dark-staining, amorphous layer that did not stain specically with aniline blue lined the cavity induced by A. pacicae. Although hatched J2 accumulated near this layer, we saw no evidence that they were feeding on it. A layer of cells, 3-to 6-cells thick, surrounded the lining of the cavity 50 DAI. Based on their afnity for stains, these cells may be more active metabolically than other cells that constitute the gall but, again, there was no evidence that the J2 fed upon them and, by 57 DAI, this layer no longer stained differentially. Certain cells in plants, known as transfer cells, are also more active metabolically than structural cells. They are characterized by dense cytoplasm and cell wall ingrowths (Gunning and Pate, 1969). Transfer cells are associated with the feeding sites of many sedentary plant-parasitic nematodes belonging to the genera Meloidogyne, Rotylenchulus and cyst nematodes, among others, but they have not been described for migratory species or those that form stem or seed galls. If the cells surrounding the cavity in galls of A. pacicae act as transfer cells to direct photosynthetic products to second-generation J2, then ultrastructural examination ( Jones and Northcote, 1972) or radiographic studies (McClure, 1977) might provide clues to their function. When mature galls were dissected in water, most of the galls contained J2 that began to move vigorously, almost immediately. Some galls, however, were lled with J2 that were rigid and stick-like. These J2 did not become active for up to an hour after release from the gall, and it was assumed that they were in a dormant state, despite the fact that the gall was moist. Secondgeneration J2 in mature galls survived desiccation and maintained their ability to infect Poa for up to 17 months. If anhydrobiosis, or dormancy, is an integral part of the life cycle, it could explain why J2 fail to emerge from the gall until it has partially decayed. Anhydrobiosis is common in other Anguinids, where it serves as a mechanism for survival and dispersal. This attribute is less critical for A. pacicae on golf course greens, where soil is seldom completely dry and the host is available year-round. However, it complicates control measures that are aimed at J2 because the occurrence of J2 in the soil would be dependent upon external factors governing the decay of galled tissues. Matricidal hatching is common in rhabditid nematodes and typical for certain entomophagous species of the genus Heterorhabditis (Johnigk and Ehlers, 1999). Adult males and J2 were observed inside living adult females of A. tritici, and the investigators concluded that the J2 hatched from eggs in the uteri of the females. They were unable to explain the presence of males (Gupta and Swarup, 1968). In older cultures, the body of an occasional adult female of A. pacicae also contained J2, but never males. Unlike this phenomenon in A. tritici, the females were always dead, and little remained of their internal organs. We examined thousands of females and never found eggs in the uterus beyond the two-cell stage. Therefore, the J2 inside the dead females of A. pacicae must have entered through the genital opening or rents in the cuticle. Anguina species are above-ground parasites that attack leaves, stems and inorescences of host plants, on which they generally form galls. Anguina graminis, a species closely related to A. pacicae, induces purplepigmented galls on the leaves of its hosts (Cid del Prado and Maggenti, 1984), but A. pacicae is the only described species that attacks only the crown. Galls were never found on the leaves proper or elsewhere on the plant. Gall color varied depending upon age of the gall and its depth in the sod prole. Galls on seedlings reared in the growth chamber were always green, a result of their exposure to bright light 12 hours daily. Galls recovered from golf course greens were white early in their development or when shielded from light by a thick layer of turf and thatch. As galls from golf greens matured, they were sometimes tinted a brownish hue, either from cellular changes within the walls of the

Biology of Anguina pacicae : McClure et al. 239 gall, or by staining from pigmented materials in the soil and decomposing thatch. On plants cultured in a growth chamber for 60 days or more, a single, secondary gall sometimes formed on the shoot above the initial gall. Because the initial gall was empty, or contained only a few second-generation J2, and the secondary galls contained developing juveniles and adults, it was concluded that secondary galls developed from J2 released from the primary gall. Both high and low temperatures limit the growth of the host, Poa annua and, consequently, that of A. pacicae. But the germination of Poa seeds from April to November on Northern California golf greens and the occurrence of secondary galls provides an opportunity for at least two generations of A. pacicae per year. When mature galls are broken and infective J2 are released by cultural practices such as mowing and aeration of greens, three or four generations would be possible. Bacteria in the cavity of the gall have been noted by other investigators (Cid del Prado Vera and Maggenti, 1984). In many Poa greens in Northern California, such bacterial galls are common, and their presence usually results in disruption of the nematodes life cycle, suggesting that the bacterium may have potential as a biological control agent. A claviform, Rathyibacter-like bacterium, that binds specically to the cuticle of infective juveniles, was isolated from galls induced by A. pacicae (results not shown). It will be the subject of future investigations. Literature on A. pacicae does not include a common name for this devastating pathogen. Because the name stem nematode is used for Ditylenchus dipsaci and because A. pacicae does not induce seed galls, the common name Pacic shoot-gall nematode is hereby proposed. LITERATURE CITED
Agrios, G. N. 1997. Plant Pathology. San Diego: Academic press. Cid del Prado Vera, I., and Maggenti, A. R. 1984. A new gall-forming species of Anguina Scopoli, 1777 (Nemata: Anguinidae) on bluegrass, Poa annua L., from the coast of California. Journal of Nematology 16:386392. Costello, L. 1983. Parasitic Nematode found in annual bluegrass greens. USGA Greens Section Record. January/February 911. Daykin, M. E., and Hussey, R. S. 1985. Pp. 3948 in K. R. Barker, C. C. Carter, and J. N. Sasser, eds. An advanced treatise on Meloidogyne, Vol. 2. Methodology. Raleigh, NC: North Carolina State University Graphics. Gunning, B. E. S., and Pate, J. S. 1969. Transfer cells - plant cells with wall ingrowths, specialized in relation to short distance transport of solutestheir occurrence, structure and development. Protoplasma. 68:107133. Gupta, P., and Swarup, P. 1968. Occurrence of living adult males and second stage larvae inside live adult females of Anguina tritici. Nematologica 14:157. Hooper, D. J., and Southey, J. F. 1978. Ditylenchus, Anguina and related genera. Pp. 7897. in J.F. Southey, ed. Plant Nematology. London. Her Majestys Stationery Ofce. Jensen, W. A. 1962. Botanical Histochemistry. San Francisco: W.H. Freeman and Company. Johnigk, S.-A., and Ehlers R. -U. 1999. Endotokia matricida in hermaphrodites of Heterorhabditis spp. and the effect of the food supply. Nematology 1:717726. Jones, F. G. W., and Jones, M. G. 1974. Pests of eld crops. New York, St. Martins Press. Jones, M. G. K., and Northcote, D. H. 1972. Multinucleate transfer cells induced in Coleus roots by the root-knot nematode, Meloidogyne arenaria. Protoplasma 75:381395. McClure, M. A. 1977. Meloidogyne incognita: A metabolic sink. Journal of Nematology 9:8890. McClure, M. A. 1988. First molt in Anguina tritici. Journal of Nematology 20:167169. Madge, D. S. 1961. The control of relative humidity with aqueous solutions of sodium hydroxide. Entomologia Experimentalis et Applicata 4:143147. Peterson, D., Winterlin, W., and Costello, L. R. 1986. Nemacur residues in turfgrass. California Agriculture 40 (March/April): 26 27. Stowell, L. 1998. Anguina pacicae seed and leaf gall nematode. PACE Insights. Vol. 4, No. 9. 4pp. San Diego, CA. Stynes, B. A., and Bird, A. F. 1982. Development of galls induced in Lolium rigidum by Anguina agrostis. Phytopathology 72:336346. Westerdahl, B. B., Harivandi, M. A., and Costello, L. R. 2005. Biology and management of nematodes on turfgrass in Northern California. USGA Greens Section Record. September-October: 7 10.

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