Journal of Nematology 18(1):112-120. 1986. © The Society of Nematologists 1986. Host Range of, and Reaction to, Subanguina picridis 1 A. K. WATSON2

Abstract: The host range of the knapweed nematode, Subanguina picridis (Kirjanova) Brzeski, under controlled environmental conditions was extended to include, in addition to Russian knap- weed, Acroptilon repens (L.) DC., plant within the Centaureinae, and Carduinae subtribes of the tribe of the family. Examination of host response to nematode infection revealed that Russian knapweed was the only highly susceptible host plant. Diffuse knapweed (Cen- taurea diffusa Lam.) was moderately susceptible, and other which formed galls were resistant to S. picridis. Key words: Acroptilon repens (Russian knapweed), biological control, gall, histopathogenesis, hy- perplasia, hypertrophy, nutritive cells, Subanguina picridis (knapweed nematode).

Most attempts in biological weed control needed to be determined before its release have involved insects and, to a lesser ex- in North America (3). The objective of this tent, plant pathogens, with few nematodes research was to examine the host range of being evaluated. The nematode Orrina the knapweed nematode, S. picridis. The phyUobia (Thorne, 1934) Brzeski, 1981, biology ofS. picridis, including its potential syn.:N0thanguina phyllobia (Thorne, 1934) as a biological control agent, and an ex- Thorne, 1961, has potential for control of amination of the of S. picridis are silverleaf nightshade, Solarium elaeagnifo- presented elsewhere (14,15). lium Cav., in the southwestern United States (11,12). The increase and artificial distri- MATERIALS AND METHODS bution of 0. phyllobia follows the inunda- Nematode inoculum: Infected host plant tive approach to biological weed control. material was obtained from O. V. Kovalev Another approach is the classical or inoc- of the Soviet Union. The material, which ulative approach whereby an exotic organ- included dried stem and pieces heavily isms's potential is evaluated for control of galled by S. picridis, was stored at room an introduced or alien weed species. A leaf temperature in a dry state until required. and stem gall forming nematode, Suban- Nematode suspensions were prepared by guina picridis (Kirjanova, 1944) Brzeski, treating galls for 3 minutes in a 1% sodium 1981, syn.: Paranguina picridis (Kirjanova, hypochlorite solution followed by three 1944) Kirjanova and Ivanova, 1968, from washes in sterile distilled water. Surface the Soviet Union parasitizes Russian knap- sterilized galls were cut open and placed weed, Acroptilon repens (L.) DC., syn.: Cen- in a 100-ml beaker containing 50 ml of taurea repens L., a major noxious weed in distilled water. The nematodes were al- much of the United States and Canada. lowed to egress from galls in continuously The nematode was reported to be host spe- aerated water overnight. Nematodes were cific and damaging to its host in the Soviet removed by pipette, suspended in distilled Union (6,9,10). Subanguina picridis galled water, and adjusted to the desired concen- Russian knapweed plants were obtained tration. from the Soviet Union. As with all other Plants tested: Plants tested included those prospective exotic biological weed control related to the known host (Russian knap- agents, the host specificity of S. picridis weed), host plants of nematode species re- lated to S. picridis, and plants which have vegetative reproduction similar to the Received for publication 26 February 1985. known host. The intensity of testing, great- 1 Research conducted at Agriculture Canada, Research Sta- tion, Regina, Saskatchewan, Canada S4P 3A2. The research est on species closely related to the known was supported by the British Columbia Cattleman's Associ- host, decreased as the test plants were more ation, the University of Saskatchewan (Hantleman Scholar- distantly related (13). Plants were grown ship), and Agriculture Canada, Regina Research Station. 2 Department of Plant Science, Macdonald College of McGill' in vermiculite in 12.5-cm-d pots and University, 21,111 Lakeshore Rd., Ste.-Anne-de-Bellevue, watered with Hoagland's solution (4) mod- Quebec, Canada H9X 1C0. I thank P. Harris for reviewing the manuscript and A. Virly ified to 10.5 ppm nitrogen. All of the ni- for preparation of photographic plates. trogen was supplied as ammonium nitrate 112 Host Specificity of Subanguina picridis: Watson 113

TABLE 1. Plant reaction to Subanguina picridis. TABLE 1. Continued.

Gall Gall forma- forma- Plants tested tion Plants tested tion Asteraceae (Compositae) Mutisieae Tubuliforae Gerbera jamesonii Bolus. Cynareae Senecioneae Centaureinae Senecio jacobaea L. Acroptilon repens (L.) DC. Vernonieae (Saskatchewan) + Stokesia laevis Greene Acroptilon repens (L.) DC. Liguiflorae (British Columbia) + acroptilon repens (L.) DC. Cichorieae (Soviet Union) + Sonchus arvensis L. Centaurea diffusa Lam. + Taraxacum officinale Weber Centaurea maculosa Lain. + Boraginaceae Centaurea ×pratensis Thuill. + Cynoglossum officinale L. Centaurea montana L. Cyanus segetum (L) Hill Caryophyllaceae Carthamus tinctorius L. Silene cucubalis Wibel Carduinae Chenopodiaceae Arctium minus (Hill) Berhn. Beta vulgaris L. Carduus nutans L. + Convolvulaceae Cirsium arvense (L.) Scop. Convolvulus arvensis L. Cirsiumflodmanii (Rydb.) Arthur + Crassulaceae Cirsium oleracum (L.) Scop. Cirsium vulgare (Savi) Ten. Sedum sp. Cynara scolymus L. + Brassicaceae (Cruciferae) Onopordum acanthium L. + Brassica napus L. Carlininae Thlaspi arvense L. vulgaris L. Euphorbiaceae Echinopinae Euphorbia esula L. ritro L, + Poaeeae (Gramineae) Anthemideae Agropyron repens (L.) Beauv. Achillea millefolium L, Avena sativa L. Artemissia gnaphalodes Nutt. Hordeum vulgate L. Chrysanthemum sp. Secale cereale L, Triticum aestivum L. Arctoteae Zea mays L. Arctotis acaulis L. Hypericaceae Gazania rigens R. Br. Hypericum perforatum L. Astereae Fabaceae (Leguminosae) Aster sp. Solidago sp. Medicago sativa L. Calenduleae Linaceae Calendula sp. Linum usitatissimum L. Malvaceae ligulistylis (A. Nels.) K. Schum. Gossypium herbaceum L. Helenieae Polygonaceae Gaillardia sp. Fagopyrum esculentum Moench Tagetes sp. Rosaceae Heliantheae Potentilla recta L. Ambrosia psilostachya DC. var. Scrophulariaceae coronopifolia (T. & G.) Tarw. Linaria vulgaris Mill. Helianthus annuus L. Solanaceae Iva axillaris Pursh Lycopersicon esculentum Mill. var, Rudbeckia sp. commune Bailey Inuleae Apiaceae (Umbelliferae) Antennaria dioica Gaertn. Daucus carota L. 114 Journal of Nematology, Volume 18, No. 1, January 1986

TABLE 2. Nematode reproduction and pathogenic reaction of plants galled by Subanguina picridis.

Reproduction Plant species within galls Degree of damage* Host ratingst

Acroptilon repens + + to + + + + Highly susceptible Centaurea diffusa + + to + + Moderately susceptible Centaurea maculosa + - to + Moderately resistant Centaurea xpratensis + - to + Moderately resistant Carduus nutans + - to + Moderately resistant Cirsiumflodmanii + - to + Moderately resistant Cynara scolymus + - to + Moderately resistant Onopordum acanthium + - to + Moderately resistant Echinops ritro - - to + Resistant Gerbera jamesonii - - to + Resistant

* Degree of damage: - = no visible damage; + = few, small galls; + + = majority of with small galls; + + + = large galls, some visible distortion of plant; + + + + = numerous large galls, plant severely distorted. 1" Host rating: resistant = few small galls, with no reproduction; moderately resistant = few to many galls with limited reproduction, but with no to very slight damage to the host; moderately susceptible = many galls with reproduction and some damage to the host; highly susceptible = numerous galls, with high rate of reproduction and severe damage to the host. and the iron in a chelate. Solutions were in one pot each at 11, 16, 20, 24, and 33 adjusted to pH 6.0 with potassium hydrox- days after nematode infestation. ide. The potting medium was saturated Galls 20-40 days old were collected. with nutrient solution every second day and Some were opened, and the contained on alternate days saturated with distilled nematodes and eggs were fixed in TAF (5). water. All experiments were conducted in The galls were then fixed, as were intact plant growth chambers at a 12-hour day galls, in formalin-acetic acid-alcohol (8). with light intensity of 1,600-2,400 ft-c at Fixed galls were washed overnight with 15+ 1Candal2-hournightat 10 + 1C. running water, dehydrated in a tertiary bu- Inoculation procedure: Each pot was in- tyl alcohol series, and embedded in paraffin fested with 1,200-1,500 nematodes sus- (7,8). Galls were sectioned at 8 ~m thick pended in 1 ml of distilled water. The with a rotary microtome and mounted on nematode suspensions were pipetted onto glass slides using Haupt's adhesive (7). Most the surface of the potting medium. Peren- sections were stained with safranin and fast nial test species, including five Russian green (2), but at least one slide of each gall knapweed control plants in each test, were was stained with haematoxylin, safranin, cut back to ground level just before infes- and fast green (7) and one was stained with tation with nematodes. Seeds of annual test periodic acid-Schiff's reagent and counter species were sown 25 days after nematode stained with fast green (7). Noninfected infestation. Each plant species was repli- leaves of the different hosts were processed cated at least five times (one plant per pot). similarly. Pots were placed in a randomized complete block design in the growth chambers. RESULTS Additional tests were conducted on Cy- Host range: In growth chambers S. picri- anus segetum (L.) Hill syn., Centaurea cyanus dis induced galls on Russian knapweed and L. (bachelor's button), an annual closely on Centaurea diffusa Lam., C. maculosa Lam., related to Russian knapweed. Five seeds C. xpratensis Thuill., Carduus nutans L., were sown (1 cm deep) in moist vermiculite Cirsium flodmanii (Rydb.) Arthur, Cynara

FIGS. 1-6. Cross sections of galls induced on Acroptilon repens, Centaurea diffusa, and Centaurea xpratensis by Subanguina picridis. (CC = central cavity, H = hairs, IN = cellular inclusions, N = nematodes, NC = nutritive cells, NEC = necrosis, PC = parenchyma cells, VB = vascular bundles.) 1) Cross section of a gall induced on Acroptilon repens. 2) Close-up of nutritive cell zone of gall on Acroptilon repens. 3) Cross section of a gall induced on Centaurea diffusa. 4) Close-up of nutritive cell zone of gall in Centaurea diffusa. 5) Cross section of a gall induced on Centaurea xpratensis. 6) Close-up of necrotic layer and cellular inclusions of gall on Centaurea x pratensis. Host Specificity of Subanguina picridis: Watson 115

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,4 116 Journal of Nematology, Volume 18, No. 1, January 1986 scolymus L., Onopordum acanthium L., Echin- formed only on leaves ofC. nutans and pro- ops ritro L., and Gerbera jamesonii (Bolus. jected almost entirely from the lower leaf (Table 1). Eggs were observed in dissected surface. The cells of the galls were not dif- galls from all the above plants with the ferentiated into definite layers (Fig. 7); the exception ofE. ritro and G. jamesonii (Table outer region consisted of elongated pali- 2). Twenty-five seedlings of the annual Cy- sade-like cells with numerous chloroplasts anus segetum exposed to approximately (Fig. 8). Most of the gall consisted of en- 75,000 nematodes failed to produce galls. larged, vacuolated, closely packed paren- Acroptilon repens: The structure and his- chyma cells with a few vascular bundles topathogenesis of S. picridis galls on Rus- partly surrounding the central cavity (Fig. sian knapweed are described in detail else- 7). Nutritive cells were not present. Some where (15). For comparison purposes, necrosis and debris were observed adjacent Figures 1 and 2 illustrate the typical highly to the central cavity. susceptible host response of Russian knap- Cirsium flodmanii: Only two small galls weed. (3 mm d) developed on leaves of C. flod- Centaurea diffusa: Galls (2-8 mm d) de- manii. Similar to most other hosts, hair de- veloped on the rachis, leaflets, and stems velopment was more pronounced on the of C. diffusa, often causing twisting and dis- gall epidermis than on the normal leaf, but tortion of infected plants. Galls were not the gall structure was not differentiated into as differentiated into layers as those on distinct zones (Fig. 9). A nutritive layer was Russian knapweed. The central cavity was not present. Some necrosis was observed often convoluted (Fig. 3). Nematodes were adjacent to the central cavity (Fig. 10). occasionally seen beyond the nutritive cell Cynara scolymus: Three small galls (4-5 zone feeding in the parenchyma cell zone mm d) developed on the leaves of C. sco- outside the vascular bundles. A smaller nu- lymus causing minor distortion of the in- tritive cell zone was present in these galls. fected leaf. The cellular layers of the galls Some necrosis was observed surrounding were not well differentiated (Fig. 11). Few the central cavity (Fig. 4). The outer layers nutritive cells were evident in some regions of the galls were similar to those on Russian of the gall (Fig. 12) but were not nearly as knapweed. extensive as in the Russian knapweed and Centaurea maculosa: Few, small galls (2- Centaurea diffusa galls. Most of the cell lay- 4 mm d) formed on leaves of C. maculosa er next to the central cavity consisted of but are not shown here. Few nutritive cells enlarged, vacuolated parenchyma cells with were present, and the gall tissue was pri- necrosis occurring adjacent to the central marily composed of enlarged vacuolated cavity. The outer layers of the gall resem- parenchyma cells. bled palisade mesophyll of the normal leaf Centaurea x pratensis: Small galls (1-4 and often had numerous chloroplasts. mm d) developed on the , leaf blade, Onopordum acanthium: Galls (3-5 mm d) and mid-vein ofC. x pratensis. A wide, dark developed on the leaves of O. acanthium staining, necrotic layer was adjacent to the with galls generally projecting from the up- central cavity (Figs. 5, 6). Nutritive cells per surface of the leaf. Galls were similar were absent from the galls which consisted in appearance to those on Cynara scolymus. largely of enlarged, vacuolated paren- The nutritive cell layer was poorly defined, chyma cells. Numerous inclusions were ev- and some necrosis was present adjacent to ident in cells adjacent to the necrotic layer the central cavity (Figs. 13, 14). The pa- (Fig. 6). renchyma cell layer extended out to the Carduus nutans: Small galls (3-5 mm d) epidermis of the gall. Cells near the epi-

..._¢ FIGS. 7-12. Cross sections of galls induced on Carduus nutans, Cirsiumflodmanii, and Cynara scolymus by Subanguina picridis. (CC = central cavity, CH = chloroplasts, EP = epidermis, H = hairs, N = nematodes, NC = nutritive cells, NEC = necrosis, PC = parenchyma, VB = vascular bundles.) 7) Cross section of a gall induced on Carduus nutans. 8) Close-up of layer of parenchyma cells with numerous chloroplasts beneath epidermis of gall on Carduus nutans. 9) Cross section of a gall induced on Cirsiumflodmanii. I0) Close-up of cell types from central cavity to epidermis of gall on Cirsiumflodmanii. 1 1) Cross section of gall induced on Cynara scolymus. 12) Close-up of cell types from central cavity to epidermis of gall on Cynara scolyrnus. Host Specificity of Subanguina picridis: Watson 117

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9 . I mm 118 Journal of Nematology, Volume 18, No. 1, January 1986

dermis were not supplied with chloroplasts ing occurred and many infected plants were (Fig. 14). severely damaged. A few infected Centau- Echinops ritro: Only one small gall de- rea diffusa plants had more than one gall veloped on E. ritro which appeared as a on a leaf, indicating that C. diffusa is mod- whitish region on the midvein of a leaf. erately susceptible to the nematode. Slight The gall was not differentiated into cell damage occurred on the other hosts, in- layers and was almost entirely composed of dicating tolerance to the parasite. Echinops closely packed, enlarged parenchyma cells ritro and Gerbera jamesonii were galled but (Figs. 15, 16). The outer layers of the gall resistant, as the nematode failed to repro- resembled mesophyll cells of the normal duce. All other plant species tested were leaf and contained numerous chloroplasts immune, as no galls were initiated, al- (Fig. 16). Nutritive cells were absent. The though most were penetrated by the nema- g.all was not circumscribed with vascular todes. tissue. Infective juveniles of S. picridis pene- Gerbera jamesonii: Four small galls (2-4 trated most plant species tested, as report- mm d) developed on one G. jamesonii plant. ed by Ivanova (6), but galls were induced The galls caused minor distortion of the on relatively few plant species. Absence of infected leaf, but nutritive cells were not gall formation in some of the Cynareae present. Extensive necrosis occurred ad- tribe species may be due to different types jacent to the small central cavity (Figs. 17, of development and growth patterns of the 18). The gall consisted almost entirely of young shoots of these plants. Two annual enlarged, vacuolated parenchyma cells with species, Cyanus segetum and Carthamus tinc- the vascular bundles laterally displaced torius L. (safflower), which are closely re- around the central cavity. lated to Russian knapweed, did not develop galls when inoculated with S. picridis. Even DISCUSSION when C. segetum was seeded at different The host range of S. picridis includes times after adding nematodes to pots to Russian knapweed and a few closely related ensure suitable meristematic tissue for in- species in the Centaureinae and Carduinae fective nematodes to penetrate, no galls subtribes of the Cynareae tribe of the As- were formed. teraceae family. Galls were induced and The histological data support the clas- reproduction ofS. picriclis occurred in plants sification of the plant species galled by S. of seven species in these two subtribes. Galls picridis as susceptible or resistant to the at- also formed on Echinops ritro and Gerbera tack of the nematode. Russian knapweed jamesonii, but the nematode failed to re- is definitely the best host that is known for produce, so they cannot be considered hosts S. picridis, and plants were severely dam- of S. picridis. aged by the nematode. The nematode in- A host rating scheme was utilized in this duced elaborate galls well supplied with study to differentiate between susceptible nutritive cells, and nematode reproduction and resistant host responses to S. picridis. was copious. Nutritive cells were reduced Resistant plants were characterized by good or absent in galls in other plants when com- host growth and poor parasite reproduc- pared to the Russian knapweed galls. Ex- tion, whereas susceptible plants were char- tensive necrosis developed in some of the acterized by poor host growth and good galls, producing a barrier between the de- parasite reproduction (1). In these studies, veloping nematodes and the nutritive cells Russian knapweed was the only plant high- or feeding regions of the galls. Although ly susceptible to S. picridis. Extensive gall- juveniles and eggs in the galls were not

FIGS. 13-18. Cross section of galls induced on Onopordum acanthium, Echinops ritro, and Gerbera jamesonii by Subanguina picridis. (CC = central cavity, CH = chlorolasts, EP = epidermis, H = hairs, N = nematodes, NC = nutritive cells, NEC = necrosis, PC = parenchyma cells, VB = vascular bundles.) 13) Cross section of gall induced on Onopordum acanthium. 14) Close-up of cell types from central cavity to epidermis of gall on Onopordum acanthium. 15) Cross section of gall induced on Echinops ritro. 16) Close-up of cell types from central cavity to epidermis of gall on Echinops ritro. 17) Cross section of gall induced on Gerbera jamesonii. 18) Close- up of cell types from central cavity to epidermis of gall on Gerbera jamesonii. Host Specificity of Subanguina picridis: Watson 119

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lmm miD 120 Journal of Nematology, Volume 18, No. l, January 1986 counted, galled plants other than Russian 8. Johansen, D. A. 1940. Plant microtechnique. knapweed supported low or no nematode New York: McGraw-Hill Book Company. 9. Kirjanova, E. S., and T. S. Ivanova. 1969. New reproduction. species ofParaguina Kirjanova, 1955 (Nematoda: Ty- ienchidae) in Tadzhikistan [in Russian]. Ushchel's LITERATURE CITED Kondara (Akademii Nauk Tadzhikskoi SSR) 2:200- 1. Dropkin, V. H., and P. E. Nelson. 1960. The 217. Translation Bureau, Canada Department of Sec- histopathology of root-knot nematode infections in retary of State. soybeans. Phytopathology 50:442-447. 10. Kovalev, O. V., L. G. Danilov, and T. S. Iva- 2. Gurr, E. 1965. The rational use of dyes in bi- nova. 1973. Method of controlling Russian knap- ology and general staining methods. London: Leon- weed [in Russian]. Opisanie Izobreteniia Kavtorsko- ard Hill. mu Svidetel'stvu Byulleten 38. Translation No. 3. Harris, P., and H. Zw/51fer. 1968. Screening of 619708, Translation Bureau, Canada Department of phytophagous insects for biological control of weeds. Secretary of State. Canadian Entomologist 100:295-303. 11. Orr, C. C., J. R. Abernathy, and E. B. Hud- 4. Hoagland, D. R., and D. I. Arnon. 1938. The speth. 1975. Nothanguina phyllobia, a nematode par- water culture method for growing plants without soil. asite of silverleaf nightshade. Plant Disease Reporter California Agriculture Experiment Station Circular 59:416-418. 357. 12. Robinson, A. F., C. C. Orr, and J. R. Aber- 5. Hooper, D.J. 1970. Handling, fixing, staining nathy. 1978. Distribution of Nothanguina phyllobia and mounting nematodes. Pp. 39-54 inJ. F. Southey, and its potential as a biological control agent for sil- ed. Laboratory methods for work with plant and soil verleaf nightshade. Journal of Nematology 10:362- nematodes. Technical Bulletin 2. Ministry of Agri- 366. culture, Fisheries & Food. London: Her Majesty's Sta- 13. Wapshere, A.J. 1974. A strategy for evalu- tionary Office. ating the safety of organisms for biological weed con- 6. Ivanova, T. S. 1966. Biological control of trol. Annals of Applied Biology 77:201-211. mountain bluet (Acroptilonpicris C.A.M.) [in Russian]. 14. Watson, A. K. 1986. MorPhological and bio- Izvestiya Akademii Nauk Tadzhikskoi SSR 2:51-63. logical parameters of the knapweed nematode, Sub- Translation No. 3793, Translation Bureau, Canada anguina picridis. Journal of Nematology, in press. Department of Secretary of State. 15. Watson, A. K. 1986. The biology of Suban- 7. Jensen, W. A. 1962. Botanical histochemistry: guina picridis, a potential biological control agent of Principles and practice. San Francisco: W. H. Free- Russian knapweed. Journal of Nematology, in press. man and Company.