Kurppa, S.

Vrain, T.C.

Name not available

Penetration and feeding behavior of Pratylenchus penetrans in strawberry roots

Kurppa, Sirpa

Vrain, Thierry C.

Jukuri

Penetration  and feeding behavior of Pratylenchus penetrans in strawberry roots Sirpa KURPPA* and Thierry C. VRAIN Agriculture  Canada Research Station, 6660 N. W Marine Drive, Vancouver, B. C., Canada V6T 1x2. SUMMARY Pratylenchus  penetrans were  inoculated  ont0  strawberry  seedlings  growing in  agar  on  microscope glass slides. The feeding  and penetration behavior of nematodes were observed for two weeks. Patterns of behavior identical to those described for other nematodes  were  recognized.  Most  nematodes  migrated to the  root  hair  zone  (widespread  exploration),  and  selected  an  epidermal ce11  by rubbing  the ce11 surfaces  with  lips  and  stylet  (local  exploration).  When  feeding,  stylet  thrusting,  salivation,  predigestion  and ingestion  phases  were  recognized.  Penetration  through  cells,  fed  upon  or  not,  occurred  after  the  nematodes  bore  a  series  of  holes in  the ce11 Wall and  forced  their  way  through.  Penetration of roots via the  root  hairs is reported. RÉSUME Comportement de Pratylenchus  penetrans durant  l'invasion des racines de fiaisier et  la prise de noum'ture Des  plantules  de  fraisier  ont  été  cultivées  sur  lame  gélosée,  de  maniere que les  racines  puissent  croître  entre  lame  et  lamelle. Le comportement  de 25 individus du nématode Pratylenchus penetrans, inoculés  près  des  racines,  a été observé durant la prise de nourriture  et  l'invasion  des  racines  pendant  près  de  deux  semaines.  Des  modes  de  comportement  similaires à ceux  décrits  pour d'autres  nématodes  parasites  ont été reconnus.  La  plupart  des  nématodes  se  sont  déplacé  vers la zone  pilifère  (attirance de loin) et  ont  choisi  une  cellule du rhizoderme ou de  l'assise  pilifère  en  explorant  la  surface  de  la  racine  par  contact  de  la  zone  labiale  ou du  stylet  (exploration  locale).  La  prise  de  nourriture  s'est  effectuée  par  l'insertion  répétée du stylet  au  travers  de  la  paroi  cellulaire, par  l'injection  de  sécrétions  digestives  dans la cellule  (salivation) et enfin  par la prédigestion  et  l'ingestion du cytoplasme  cellulaire. Les nématodes  ont  pénétré  dans  les  cellules,  dont  ils  s'étaient  nourris  ou  non,  en  perçant  avec  leur  stylet  une  série  de  trous  alignés, affaiblissant  ainsi  une  des  parois  cellulaires et y forçant un passage.  Certains  nématodes  ont  pénétré  dans  les  racines  en  passant par  les  poils  absorbants. The feeding behavior of migratory endoparasitic nematodes is poorly documented relatively to that of ectoparasites (Doncaster, 1971 ; Wyss,  1981). Doncaster and Seymour (1973) analyzed the behavior of eleven species  of Tylenchida. They systematized the different stages of exploration, penetration and feeding, and presented a set of assumptions to explain the observed behavior of these nematodes. Whereas the movements of Pratylenchus species are difficult to follow inside of roots, their histopathology, namely the formation of lesions,  is  easily seen and well documented (Townshend, 1963a, 1963b; Troll & Rohde, 1966; Mamiya, 1970; Acedo & Rohde, 1971; Corbett, 1972; Pinochet, 1978). Pratylenchus  penetrans (Cobb) Filip. & Sch. Stek. prefers to enter roots in the region of root hair develop- ment, although the zone of elongation can also be invaded (Troll & Rohde, 1966; Townshend, 1978). P. penetrans feeds  mainly on the cortical cells, where cavities are  formed  when the tissue collapses. In most plant  host roots  these cavities can  be seen à few  hours to a few days after penetration as  discrete  brownish lesions of varying sizes (Townshend, 1963b; Acedo & Rohde, 1970), but not in cereals (Troll & Rohde,  1966). The object of this  study was to observe the penetration and feeding processes of P. penetrans in strawberry roots. Materials  and  methods Small  strawberry plantlets, cv. U. C. 11, were soaked for 1 min in 0.5 O/o sodium hypochlorite solution and rinsed three  times in sterile water. Thin layers  of  0.8 O/O water  agar  containing two drops of Hoagland  solution were poured over glass slides  (25 x 82 mm) and covered partially with a cover slip (22 x 40 mm). One plantlet * Present  address : Agricultural  Research  Center, 31600 - Jokioinen,  Finland. Revue Nématol., 8 (3) : 273-276 (1985) 273 S. Kzuppa & T C. Vrain was placed ont0  the agar at  the  uncovered  end of each glass slide. The plantlets on the slides were kept in a  petri dish on a  moist  filter  paper at 25O, under fluorescent tubes with daylight of 16 h and light intensity of 37 p Einstein m-'s-'. The slides  were set vertically so that  the roots grew underneath  the coverslip. Al1 stages of Pratylenchus penetrans, extracted  from infected  raspberry roots, were used  for  inoculum. The nematodes were axenized in  72  hours in a 4 ppm solution of methoxy ethyl mercury  chloride  (Aretan @), with  sterile  air  bubbling  through the solution  (modified from  Townshend, 1963a). When the strawberry  plantlets had grown two to three roots, 1 cm in length, twenty sterile  nematodes were handpicked  and placed near  the roots.  Strawberry  plantlets were also grown in  sand in 20 ml polystyrene microbeakers and inoculated to  study histopathology. For  the behavioral study, 25 nematodes  on or inside of roots were observed for two weeks at 400 or 1,000 x magnification  without  precautions to keep the preparations sterile. For the ultrastructural study, the slides with  plants  and agar, and pieces of roots  from  the inoculated  plants growing in  sand, were placed in  liquid nitrogen. The frozen roots were placed in plastic  tubes (BEEM@ embedding capsules, size 00) 13 mm long, 8 mm diam., closed at the bottom with 15 pm pores nylon sieve. The roots were fiied  in 4 O/O glutaraldehyde in 0.05 M sodium cacodylate buffer  (pH 6.8) for 12  hours. The roots growing in agar were placed in warm 0.05 M cacodylate buffer  to remove the agar. Al1 roots were post fiied in  2 O/O osmium  tetroxide  for two hours and  dehydrated in  an ethanol serie starting with 20 O/o ethanol. When in 70 O/O ethanol the roots were examined under a  stereomicroscope  and pieces 1-2 mm long were selected  for  further  dehydration a d embedding in  Epon 812 @ or, critically point  dried  for SEM.  The embedded material was cut  into 0.5 to 1.5 Pm thick sections, with an ultramicrotome  and  stained  with  a  polychrome  stain (Van Reempts & Borgers, 1975). Observations  and  results Most nematodes moved to the root  hair  region  within three hours of being transferred to the agar. They explored  the  root by touching the surface of epidermal cells with  their lips, and  protracting  their  stylet  enough to  touch  but  not t  Pierce the ce11 walls. Some  nematodes explored  root  hairs  along  their  entire  length. Very sel- dom were they  seen  exploring  a  root tip or ce11 elonga- tion  zone and these areas were never fed upon or penetrated. Following local exploration of ce11 surfaces  the  area beside an intercellular Wall would  be pierced, and  the stylet  thrusted several times  into the cell. The epidermis ce11 walls appeared very elastic and difficult  to  puncture as many  nematodes  had  to  repeatedly thrust their  stylet 274 to get through. For their initial feeding, before pene- tration, some nematodes  did  not  puncture  an  epidermal ce11 Wall but instead  pushed  their  stylet intercellularly to the cortical ce11 beyond.  Traces of feeding  on  epider- mal cells, i.e. the minute holes made through the ce11 walls,  were difficult to recognize with certainty  in SEM or semi-thin  sections. Root  hairs were sometimes  penetrated but never fed upon. The nematodes  would Pierce the ce11 walls seven to  ten  times  along  a line, then  tear  a hole (Fig. 1A) by pressing  their  head and waving it vigorously until it was inside the root hair. Sometimes they would drawback and  open  another hole on  the  opposite side  of the hair. The cytoplasm of these cells circulated vigorously du- ring and after nematode penetration, then the cells would collapse rapidly. Several nematodes penetrated the  roots via the root  hairs,  through the basal epidermal cells to  the  cortical  tissue. Once  their  stylet was inside an epidermal or a cortical cell, the  nematodes  stopped moving. During salivation the stylet was sometimes reorientated within the cell. Their median  bulb  pulsated only two to three times in 5 or 6 seconds, a globular secretion was seen passing from  the stylet tip  into  the cell, and  the movement of the cytoplasm became more rapid. The nematodes were inactive for 2 to 5 mn before ingestion began. Their median bulb contracted  a few times in  the next 2 mn, and  then  pulsated  at several contractions per second for 7 to 8 mn. There was only one salivation and one ingestion  period in  the epidermal cells, before the nematodes  penetrated (Fig. 1B). Several nematodes  would  often  penetrate  neighboring epidermal cells or  even  go  through the same hole in an epidermal cell. The nematodes moved through  epider- mal cells to cortical cells centripetally,  and once in  the cortex they migrated along the root length, moving slowly from ce11 to cell, making every time  a row  of holes and  pushing  their  head through  into  the adjacent cells. The cavities made  by the collapse of cells fed  upon  and the  penetration of the nematodes were quickly colonized by bacteria (Fig. 1C and D). Al1 the  nematodes under observation remained inside the  roots  for the  duration of the experiments  and  many eggs  were laid in  the cortex. In  the injured cortical cells the  intercellular  areas were enlarged and intensely colo- red in blue in  the polychrome  stained  semi-thin sections, whereas uninvaded  areas of the cortex  did  not  take any stain. In  the two weeks  of the  experiment  the  nematodes did  not  penetrate the endodermis,  and  did  not  migrate to  the zone of differentiation, the root  tip area, or  the zone of initation of lateral roots. Discussion The behavior of Pratylenchzcs penetrans follows the pattern  recognized for most  other  nematodes  (Doncas- Revue Nématol., 8 (3) : 273-276 (1985) Penetration  and feeding behavior of Pratylenchus  penetrans Fig. 1. A : penetration  hole in root  hair  of  strawberry  after Pratylenchus  penetrans entered  the  root  and  the  root  hair ce11 has  collapsed. Arrow  shows  bacteria; B : nematode  penetrating  epidermal  cell; C : penetration  hole in epidermal ce11 after Pratylenchus  penetrans entered the root;  numerous  bacteria  are  found  near  and in the opening; D : cavity  formed  by Pratylenchus  penetrans in  cortex  of strawberry  root; the cavity  is  rapidly  colonized  by  bacteria. (A, B, C : SEM; bar = 5 Fm). Revue Nématol., 8 (3) : 273-276 (1985) 275 S. I<urppa & T. C. Vrain ter, 1973; Wyss,  1981). The nematodes  migrated  almost exclusively to  the  root  hair  zone  to  feed  and  penetrate (widespread  exploration).  Once in the  feeding  zone of the root  they  selected an epidermal ce11 or a  root  hair and probed the ce11 surface with lips and stylet to find a suitable  penetration  site (local exploration). The nema- todes were somewhat immobile while injecting their saliva which appeared as opaque globules similar to those  described in other  nematodes (Wyss,  1981). If  we assume that  the role of the saliva is to lower the viscosity (predigestion) of the cytoplasmic material to  be ingested, then  the few slow contractions of the  median bulb preceding the  tiue ingestion period might  indicate a monitoring of the external digestion process by the nematode.  Whether the ce11 content was ingested  or  not, a series of stylet thrust  punctured  and weakened the ce11 Wall along  a  line  through which the  nematodes  forced its way into  the cell,  as described  for Pratylenchus crenatus (Klinkenberg, 1963), and Helicotylenchus  dihys- tera (Jones, 1978). Temperature influences the speed of feeding and penetration  behavior  (Townshend, 1978,  Boag,  1980). In Our experiment the speed of stylet  thrusting  and  meta- corpus  pulsations  could  not  be analyzed accurately. The observations were not done at a  constant  temperature under  the  microscope  and  the  data given are averages of many  observations. Penetration of roots via the  root  hairs has not  been reported for other endoparasites. However, although observed several times, it did  not  constitute  a  signifïcant route  to  the  cortex;  penetration  through  epidermal cells was more  common.  Feeding  on the endodermis was not observed in  the two weeks of  Our experiments,  although others (Acedo & Rohde, 1971 ; Mamiya, 1971; Corbett, 1972;  Pinochet,  1978) have shown that Prarylenchus spp. will eventually  migrate  to  and  feed  on  this  tissue. REFERENCES ACEDO, J. R., & ROHDE, R. A., (1971). Histochemical root pathology of Brassica oleracea capitata L. infected  by Praty- lenchzts penetrans (Cobb)  Filipjev  and  Schuurmans  Stekho- ven  (Nematoda : Tylenchidae). J. Nematol., 3 : 62-68. BOAG, B. (1980).  Effects  of  temperature  on  rate  of  feeding  of 'the  plant  parasitic  nematodes Rotylenchus  robustus, Xiphi- Accepté  pour  publication le 22 mai 1985. nema  diversîcaudatztm and Hemicycliophora  conida. J .  Ne- matol., 12 : 193-195. CORBETT,  D.  C.  M.  (1972). The effect  of Pratylenchus fallax on wheat, barley and sugarbeet roots. Nematologica, 18 : DONCASTER,  C.  C.  (1971).  Feeding i  plant  parasitic  nemato- des. Mechanisms and behavior. In : Zuckerman, B. M. & Rohde, R. A. (Eds) : Plant  Parasitic  Nematodes, Vol. II, New York,  Academic  Press : 137-157. DONCASTER,  C.  C. & Seymour, M. K., (1973).  Exploration  and selection of penetration site by Tylenchida. Nematologica, JONES, R. K. (1978). The feeding  behavior  of Helicotylenchus spp.  on  wheat roots. Nematologica, 24 : 88-94. KLINKENBERG,  C.  M.  1963.  Observations  on  the  feeding  habits of Rotylenchus uniformis, Pratylenchzts crenatus, P. pene- trans,  Tylenchorhynchus  dubius and Hemicycliophora  similis. Nematologica, 9 : 502-506. MAMIYA, Y. (1970). [Parasitism and damage of Pratylenchus penetrans to Cryptomeria seedlings.] J. Jap. For. Soc., 52 : PINOCHET, J. (1978).  Histopathology  of the root  lesion  nema- tode Pratylenchus  coffeae on  plantains,  Musa  AAB. Nemato- logica, 24 : 337-340. TOWNSHEND, J. L. (1962). The root lesion  nematode Pratylen- chus penetrans (Cobb,  1917)  Filip. & Stek.  1941,  in  celery. Can. J .  Pl. Sci., 42 : 314-322. , TOWNSHEND, J. L. (1963a). The pathogenicity of Pratylenchzts penetrans to celery. Gan. J. Pl. Sci,  43 : 70-74. TOWNSHEND, J. L. (1963b). The pathogenicity  of Pratylenchzts penetrans to strawberry. Gan. J .  Pl. Sci., 43 : 75-78. TOWNSHEND, J. L. (1978).  Infectivity  of Pratylenchus  penetrans on  alfalfa. J.  Nematol., 10 : 318-323. TROLL, J. & ROHDE,  R.  A.  (1966).  Pathogenicity  of Pratylen- chus  penetrans and Tylenchorhynchus claytonion turfgrasses. Phytopathology, 56 : 995-998. VAN  REEMPTS, J., & BORGERS, M. (1975).  A  simple  polychrome stain  for  conventionally fîîed epon  embedded  tissue. Stain Technol., 50 : 19-24. WYSS, V. (1981).  Ectoparasitic  root  nematodes : Feeding behavior  and  plant ce11 responses. In : Zuckerman, B. M. & Rohde, R. A. (Eds) : Plant Parasitic Nematodes, Vol. III. New  York,  Academic  Press : 325-321. 303-308. 19 : 137-145. 41-50. 276 

Penetration and feeding behavior of Pratylenchus penetrans in strawberry roots

Abstract

Similar works

Full text

Available Versions

Name not available

Jukuri