host status differences and their relevance to damage by ... › 309e ›...

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Nematology, 1999, Vol. 1(4), 421-432 1'5s A/ .p"P3ycg - 44"qq-

Host status differences and their relevance to damage by Meloìdogyne incognita

Mahmoud E. EHWAETI Mireille ARGETTE 2, Mark S. PHILLIPS and David L. TRUDGILL '**

BP504.5, 34032 Montpellier; Fraiice Nertiatology Departineut, Scottish Crop Research Institute, Iiivergowrie Diindee DO2 5DA, Scotland; ORSTOM.

Accepted for publication: 29 April 1999

P Summary - Four populations of Meloidogjrie incognita were tested on 57 species of plants from temperate and tropical regions. Juveniles of one or more populations invaded, and induced galls on almost all plants. Except for the population from the West Indies which induced most galls on Nicotiana spp., there was no obvious relationship between the origins of populations n'nd their ability to induce galls on plants from particular regions. Although most plants were hosts for all four populations, many produced only a fen galldegg masse; with some of the populations. The differences in galling between populations, and the interaction with plant species were both significant ( P 4 0.001). Poor hosts often contained fewer juveniles and the rate of juvenile development was often decreased compared with good hosts. Solamim nigrilin was an exception in that it was invaded by fewer juveniles than susceptible tomato, but rates of development were similar. The effect of host status differences in four plant species on damage by M. incognita was investigated but only the growth of susceptible tomato was decreased. The growth of the non-host Urtica dioica, of the poor host Datiira nietal, and of an intermediate host S. nigruni, was unaffected.

Zusammenfassung - Unterschiede im Wirtsstatiis und ihre Bedelitring fiìr den durch Meloidogyne incognita veriirsacliteii Schaden - Vier Populationen von Meloidogyne incognita wurden an 57 Pflanzenarten aus gemässigten und tropischen Gebieten gepriift. Juvenile einer oder mehrerer Populationen wanderten in fast alle Pflanzen ein und induzierten Gallen. Mit Ausnahme der Population von den Westindischen Inseln, die an Nicotiana spp. die meisten Gallen induzierte, bestand keine deutliche Beziehung zwischen der Herkunft der Populationen und ihrer Fähigkeit, an Pflanzen bestimmter Gebiete Gallbildung zu induzieren. Obwohl die meisten Pflanzen Wirte für alle vier Populationen waren, bildeten viele von ihnen mit einigen Populationen nur wenige GallenEimassen. Die Unterschiede zwischen den Populationen in Bezug auf Gallbildung und auf Wechselwirkungen mit den Pflanzen waren beide significant ( P < 0.001 ). Schlechte Wirte enthielten oft weniger Juvenile als gute Wirte, und die Entwicklungsrate der Juvenilen war oft vermindert im Vergleich zu guten Wirten. Solantrrii nigrurii bildete insofern eine Ausnahme, als weniger Juvenile eindrangen als in anfallige Tomaten, die Entwicklungsraten aber ähnlich waren. Die Wirkung der Unterschiede im Wirtsstatus auf die Schädigung durch M. incognita wurde an vier Pflanzenarten untersucht. Nur das Wachstum von anfälligen Tomaten wurde vermindert. Das Wachstum des Nichtwirtes Urtica dioica, des schlechten Wirtes Datura rnetnl und des mässig guten Wirtes S. ~ i i g r i i i ~ wurde nicht beeinflusst. Keywords - development, fecundity, host range, immune hosts, invasion, poor hosts, reproduction, resistance, root-knot tolerance, tomato, weed hosts.

I

The species of Meloidogyiie which reproduce by mitotic parthenogenesis (e.g. M. incognira) have exception- ally wide host ranges (Jepson, 1987) compared with other biotrophic pathogens. Trudgill (1 997) suggested that mitotic parthenogenesis was a means of conserving a genome which conferred a wide host range by largely preventing Co-evolution with particular groups of plants. Con- sequently, although some plant species are good hosts, which support rapid and large increases in population density, many others are relatively poor hosts.

It is uncertain whether poor host status and resistance are usually analogous and involve similar genes and mechanisms, especially when considering planthematode interactions involving mitotically parthenogenetic species such as M. incognita (Blok et al., 1997; Trudgill, 1997). However, Anwar et al. (1994) found that rates of root invasion and of juvenile development by M. incognita were less in poor than good hosts. Similarly, with two cultivars of carrot (Dacrciu carota) considered partially resistant, Huang (1986) observed that fewer juveniles of M. ja-

* Corresponding author, e-mail: dtrudg@ scri.snri.ac.uk

@ Koninklijke Brill NY Leiden, I999 47 1

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Iyrizica invaded, and in one cultivar they developed more slowly than in a more susceptible cultivar. Also, the resistant cultivars were less damaged than the susceptible cultivar. Bird (1972) observed that invasion, development and fecundity of M. jaimiica were all less on bean (Vicia fclba) than on tongo (Lycopersicoii escr~leiituin) and Fer- ris et al. (1984) observed differences i n rates of egg production by M. areimrin on grapevine cultivars of differing host status.

The effects on plant growth of differences i n host status are potentially further compounded if time allows for several nematode generations during the growing season. Ehwaeti et al. (1998b) showed that a low, initially non- damaging population of M. incogriita could increase so greatly that susceptible tomato (L. escz~leiztu~iz cv. Mon- eymaker) became damaged later in the growing season. Consequently, it seems probable that poor hosts are likely to be less damaged (i.e. appear more tolerant) than good hosts because invasion, and hence initial damage and fecundity, and damage later in the growing season, are decreased.

This paper presents the results of four experiments with the following objectives:

1. To test the hypothesis that the ability to parasitise a wide range of plants is inherent to M. iiicogizita, and has not arisen as a process of Co-evolution with each host species. This was examined in two experiments by comparing the host status to four populations of M. iizcogizita from different parts of the world of a range of wild plants from different families and regions, including temperate areas. These experiments also tested the converse hypothesis i.e. that non-host status/resistance does not necessar- ily arise through a process of CO-evolution.

2. To examine the mechanisms involved in host status differences by assessing the relationship between host status and numbers of nematodes invading and their subse- quent rates of development and fecundity.

3. To test the hypothesis that host status affects tolerance of damage by M. iizcogiiitn by comparing the effects of increasing inoculum density on the growth of four plant species of different host status.

Materials and methods

Four single egg mass lines ( L l j originally from Thai- land, L19 from Martinique, L20 from French Guiana, and L27 from North Carolina) identified as M. iizcogizita by isozyme electrophoresis and by molecular DNA analysis (Blok, pers. comm.), were used. All four lines had

been maintained for several years on susceptible tomato cv. Moneymaker in a glasshouse and were avirulent on resistant tomato cv. Rosso1 (see Fargette et al., 1996). To produce the inoculum, each line (hereafter referred to as a population) was sub-cultured for 60 days in sterilised soil on tomato cv. Moneymaker in a glasshouse at about 24°C. An egg inoculum was produced using NaOCl for extraction as described by Ehwaeti et al. (199%). Freshly hatched juveniles (52) were obtained from roots with egg masses placed in a mist chamber.

Seeds of wild plants from a temperate region, were supplied by the Department of Animal and Plant Sci- ence, University of Sheffield. Seed from tropical and sub- tropical species within four major Families (Solanaceae, Leguminoseae, Chenopodiaceae, Compositae) were supplied by the Kew Seed Bank Collection, Royal Botanic Gardens, Bolney. Other seed came from the collection maintained at the Scottish Crop Research Institute.

Four experiments were conducted in a glasshouse with set temperatures of 22°C at night and 26°C during the day-time and with supplementary lighting to extend the day length to 14 h when needed. Nutrients were supplied as liquid feed to ensure plants did not become nutrient deficient. The experimental blocks were re-randomised and rotated at regular intervals to try and provide a similar environment for each plant.

ASSESSMENT OF HOST STATUS TO FOUR POPULATIONS OF hf. INCOGNITA

Experiineiit I Seeds of 24 species, mainly of wild plants from the

UK collected by Sheffield University, but some from seed stocks held at the Scottish Crop Research Institute (SCRI), were tested as hosts for the four populations of M. iizcogizita (Table 1). The seeds were germinated in sterile soil and then transplanted, one seedling per pot, into 7 cm diam. pots filled with sterilised peadsand mixture. The smallest seeds were sown and transplanted 2 weeks before the larger seeds to try and establish plants of similar size at inoculation. This was not entirely successful, and 2 or 4 weeks after transplanting the plant species were arranged into three blocks comprising the largest, medium sized or smallest plants. Susceptible tomato (L. esculeizfuiii) cv. Moneymaker plants of a similar size to the test plants were used as susceptible controls for each block. Each plant was inoculated with about 500 eggs pipetted into three holes made in the soil at its base. Three replications of each host and population combination were used and after 42 days the roots were washed free from soil, weighed

122 Neiizatolopv

Meloidogyne host statics arid damage

Table 1. Nuinbers of palls produced (iiieaiis of 3 replicates) by four popdarioris of Me1oidog)me incognita oii 24 species riiairilj ofll.i[d plants fioiiz the UK ordered by overall mean. Three sizes of toiiiato plants were used as susceptible controls.

GenusISpecies Root weight (g) Number of galls/plant

Population Plant mean 1)

L15 L19 L20 L27

Trifoliiíiii reperis Epilobiuiiz liirsirtiiiiz Dacicils carota Datura straiizoiiiirni Petiinia spp. Silene dioica Medicago hrpuliria Solaiiiciii iiigriiiiz .-. Clzeriopodiirnz horale Spinacia oleracea Cai?lf;aliirla rotilridifolin Pasf;:iiaca sativa LO& coriiicir~atiís Areiiaria serpyllifolia Achillea rizillefoliiciiz Hieraciuiiz pilosella GonipliTeiin spp. Cerasríp foiifaiiiriiz Leoiitodp hispidits Dahlia spp. Eriopho~ciiz vagiiiaturii Capsicimi) a~ziiii~ciiz Corzyza calladeilsis Urtica diiica

.4%-

4

LSD4) /i

PopulatiÓn mean gall number

LSD !i

Lycopersicoiz esculeiitiíiiz (1)') 12.5 Lycopersicon esciileiituiii (in) 11.2 Lycopersicon esculentitiiz (s) 10.2

2.2 7.1 5.0 6.1 2.2 7.3 2.4 3.3

~ 3: .. 1 .o 1.1 2.0 7.9 0.8 2.3 5.2 6.7 6.4 2.7 2.7 5.5 0.3

13.5 5.9 6.4

,..- <:

1.58

90.0 80.0 76.7 86.7 93.3 76.7 32.3 30.7

1.7 28.3 17.0 %.O3) 3.0 30.03) 1 .o 18.0 9.0 30.0

i 3.7 17.3

29.33) 4.0

9.3 13.0 6.7 22.0 6.7 13.7 7.0 9.7

20.0 9.0 0.0 15.3

12.7 5.7 3.7 9.3 7.0 2.3 8.0 2.0 0.67 3.0 0.0 ' 2.3 0.0 . ' 0.0 0.0 0.0

53.33) 3.7

25.03) 3.7

' 3.38

21.00 71.16

17.53

100.03) 93.3 83.3 48.3 56.73) 44.03) 45.03)

21.0 17.3 3.0

14.0 25.73) 17.0 6.3

13.3 22.3 5.0 4.0 4.3 8.7 6.7 6.0 3.3 3.7 0.0 0.0

53.33)

26.16

80.0 63.3 '

53.3 40.0 30.7 22.0 11.0 6.7

16.3 7.7 0.7

10.7 I .7 9.3

13.7 12.0 6.3 2.3

10.3 3.3 3.7 3.7 0.0 3.0 0.0 0.0 0.0

15.25

81.5 80.0 76.7 37.8 36.8 34.6 22.3 19.8 19.1 16.5 15.2 14.5 14.0 12.3 12.2 11.4 11.3 9.1 7.4 6.5 6.3 4.9 4.0 2.5 1.5 0.0 0.0

8.7

a 1) ~ $ í í of 12 plants. 2, (l)y= large, (m) = medium, (s) = small plants. 3, Sïgnificantly ( P < 0.05) more galls than that with at least two of the other populations. 4! LSD, least significant difference at P -= 0.05. li ? and the numbers of galls counted and the presence of egg masses noted.

Experiineizt 2

Seeds of 34 species from mainly sub-tropical and tropical regions supplied by the Royal Botanic Gardens, Kew, but some from the SCRI collection of virus indicator

plants, were grown, tested and assayed as for Experi- ment 1. Pots (three replicates for each plant nematode

--,combination) were inoculated with 500 eggs of popula- 5, L19 and L20, but with only 300 eggs/pot of n L27. The origins of the plants are listed in Ta-

ble 2. Where possible, species within a genus came from

'1.

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Vol. 1(4), 1999 423

M.E. Eliwaetì et al.

Table 2. Nuiiibers of galls(iiieaiis of 3 replicates) produced by foiìr populatìoiis of Meloidogyne incognita on 32 species iiiaìiily of wild plciiitsfr.oiiz tropìcal/sub tiupical regions ordered by overall iiieaii. Susceptible toiiiato MYIS used as the coiitrol.

GenuslSpecies Origin Root weight (g) Number of gal¡s/plant Population Plant meaiil)

L15 L19 L20 L27

Lycopersicori esculeiituiit2) í'Ì-ifolì~riii usaiiibareiise Nicotiaria occidentalis2) Nìcoriaiin xaiztlii2) So1a1111111 Illallllllosu111

Clieiiopodiuiit album Clieiiopodiiriii strìctuiii ~rifo1ìiriiz repens2) Nicotiaiin debiieyìi2) Nicotiaiia gluriiiosa2) Clieiiopodiuiiz iiiurale ~~ieiiopodiiriii aiiiaraiztìcoloi2) Nicoriaiia tabacLiin*) Solaiiiriiz sodaiine~riiz Solaiiiriii iiicariuiit Wiiirliaiiia soimifera Cheiiopodiiiiiz alburiz ~/?eiiopo~iiriit qiiiiioa2) S O ~ ~ ~ I i t ~ i i Z viarwiz Goiiiplireiia globosa Clieiiopodiuiii capitatuiii Melilotiis indica PIiysalisf(oridaiia Darir in inetel Spinocia iiiedaiiìa Nicotiaim beiitkaiiziaiia2) Darirra iiinoxia Medicago sativa Clieiiopodiiriiz iiiurale Melilotirs albus Darirra fer0.v Seiiecio loiigilobiis Leoiitodoii taraxacoides Seiiecio clivìcola

N. America Africa N. America N. America S. America N. America Asia Other N. America ,

N. America Other Other N. America Africa Africa Africa Africa Other Asia N. America N. America S. America N. America Africa Other N. America Africa N. America Africa N. America Africa N. America Asia S. America

11.0 100.0 100.0 81.7 70.0 87.9 1 .o 71.73) 36.0 61.73) 26.0 48.8 1.8 64.03) 96.73) 11.0 10.7 45.6 6.4 26.7 100.0~) 30.0 25.0 45.4 6.1 71.7 52.3 43.3 8.0 43.8 2.4 47.0 34.3 31.0 13.3 31.9 1

4.4 38.3 33.0 38.3 11.7 30.3 1.3 20.0 35.0 31.7 20.3 26.8 2.3 33.3 36.0 30.0 7.0 26.6

1.4 44.73) 43.33) 12.0 3.0 25.8 7.7 35.0 49.3 10.0 5.3 24.9 7.2 21.7 50.03) 7.7 16.7 24.0 4.7 30.7 31.7 20.0 0.0 20.6 3.7 31.7 25.0 21.7 3.7 20.5 5.2 25.0 5.3 22.0 25.0 19.3 0.6 47.73) 11.0 9.0 2.0 17.4 7.5 5.3 37.33) 11.7 6.3 15.2 5.4 43.33) 12.0 0.7 0.7 14.2 4.1 15.0 21.7 12.0 4.3 13.3 0.8 3.0 30.33) 14.0 2.7 12.5 0.6 9.3 22.3 6.3 3.5 10.4 9.0 27.33) 4.0 2.0 1 .o 8.8 8.8 3.7 15.0 10.0 5.0 8.5 1.9 14.7 3.0 6.0 2.0 6.6 3.2 4.3 5.8 4.8 4.7 4.9 9.9 3.0, 8.7 5.3 1.3 . 4.6

4.4 0.5 5.3 5.7 3.0 2.0 4.0 I

1.9 2.3 6.7 0.7 1.3 2.8 7.4 2.0 3.0 3.0 1 .o 2.3 2.6 2.3 1.7 1.5 0.3 1.5 4.1 3.0 0.7 0.0 0.0 0.9 6.9 0.0 0.0 0.0 0.0 0.0

3.9 16.7 55.03) 13.3 21.0 26.5 I

3.1 10.0 3.0 3.7 1.0 *

1.22 3.10 9.32

Population mean gall number 25.2 27.8 15.6 8.8 I

LSD 18.62

' ) Mean of 12 plants. 2, From SCRI seed collection. 3, Significantly ( P < 0.05) more galls than that produced by at least two other populations. 4, LSD, least significant difference at P < 0.05.

424 Nematology

Meloidogyne host statzis arid daiiiage

Table 3. Total root leiigtlz and izicitzbers of root tips aiid iizemi iiiiriibers of juveniles (J2) of Meloidogyne incognita (popirla- fions LI5 arid L20) per root-tip 5 days afier iiiociilarioii.

Seeds were germinated in Petri dishes on a damp filter paper. Species with small seed were sown and planted 1 week before those with larger seed to produce similar sized plants at inoculation. One 4 day-oid seedling was

root No root No J2 transplanted into each tube and allowed to grow for 3 days (big seed) or 10 days (small seed) when all the tubes

Host status Plant species length (cm) tips per root

- Good Lycopersicori esciilerztrrnz 3.52 7.5 4.19 cv. Moneymaker

Solalzzlllt lnaml~zosuln~) 3.10 7.6 3.50 Trifoli1ci7z repens 1.80 2.6 6.15

Good to moderate

Poor - Achillea millefoliziitz 1.72 2.8 3:57 Solaiiiiiii izigruiii 2.07 4.7 1,!74 -_ - Non

Coiiyza caiiadensis Y..? 1.64 1.9 0.00 ~eneciq'clivico~a ) 1.10 4.2 0.86 Urtica&ìioica 0.81 1.4 0.57

L. escÙleiitui7z cv. Rossol]) 3.40 7.0 0.37

LSD (P = 5%) 0.65 1.94 2.5

Resista&

Tested only with population L20. Other values are means of results for pdpulations LI5 and L20 combined.

were inoculated simultaneously. A shortage of inoculum and seeds of some species meant that Solaniiin clivicola, S. inaininosuin, and resistant tomato cv. Rossol were not inoculated with population L15. There were 30 replicates for each plant species and nematode combination.

The planted tubes were fully randomised in a water bath at 25°C and watered daily as required and fertilised once each week with 5 ml per tube of diluted Vitafeed-III (N19%, P2O5 19%, K20 19%). Five tubes of each treatment were removed at 5, 15, and 35 days after inoculation. The roots were gently washed free of sand by plac-

Care was taken not to damage or lose the root tips. Any juveniles or males in the sand were collected for counting by pouring the washing water through 75 ,um and 53 ,um sieves and then placing the sievings on a Baermann funnel for 24 h. Root length and numbers of root tips and of galls were determined and the entire root system was stained with acid fuchsin. Root systems were pressed between glass plates (7.5 x 5.0 cm) and the numbers of nematodes and their stage of development were determined un-

I

I ing them in a bucket and rinsing with a gentle jet of water. I

der a stereo-microscope. At 35 days the egg masses were stained with phloxine B (Holbrook et al., 1983) and eggs released for counting by dipping the roots in 1% NaOCl

different coniinents. Tomato cv. Moneymaker was used as a control as for Experiment 1.

>'

EFFECTS 0N:INVASION AND DEVELOPMENT I

Experiment 3 The relationship between host status and rates of ju-

venile invasjbn, development and fecundity were studied with seedlifigs (2 to 3 cin high) growing in 2.5 x 15 cm glass boiljng tubes containing steam-sterilised sand. Each tube was!inoculated, using a hypodermic syringe, with 50 (& 14) freshly hatched J2 from one of two populations of M. inc&nita (L15 or L20). The plants tested were selected main$ based on the results from Experiments 1 and 2 (see Tables 1 and 2). They were three putative non-host species ,(Senecio clivicola, Urtica dioica, Conyza canadensis), a resistant host (tomato cv. Rossol, which contains the Mi gene), two poor hosts (Achillea inillefolium, and Solanuin nignim which was a poor host for L20 but a good host for L15), and two good to moderate hosts for both populations (Solanum inaïniizosuin, Trifolium repens). Tomato cv. Moneymaker was used as a susceptible control.

..

for 10 minutes (Hussey & Barker, 1973).

HOST' STATUS A N D DAMAGE

Expeiiinent 4 Four plant species (U. dioica, Datura metel, S. izigiunz,

and susceptible tomato cv. Moneymaker) were chosen, on the basis of the results in Experiments 1 and 2, to provide a range spanning from a non to a good host. Because of their relatively small size, seeds of U. dioica were broadcast- sown in small trays containing sterilised potting compost in a glasshouse 1 month earlier than those of the other three species. Eight weeks later (4 weeks for D. inetel, S. izigr~iin, L. esculei1tunz) two seedlings were transplanted i>b a 15 cm diam. pot filled with 2 kg sterilised soil (40% peath6Q% sand).

Duril;'@f@ng, each pot was inoculated with 110 ml of egg suspension, successively mixed by hand into the bot- tom, middle and top layers to try ensure the eggs were distributed throughout the soil. Three densities of M. iizcog-

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I l: Vol. 1(4), 1999 425

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M.E. El~ivaeti et al.

Table 4. Nuiilbers of iieiiiarodes per plant (iizeaiis of 5 replicates) at 5, 15 arid 35 days aper ii7oculatioii with two populatioiis of Meloidogyne incognita

After 5 days After 15 days After 35 days Host status Population Population Population

Plant species L20 L 1.5 L20 L15 L20 L15

- Good Lycopersicoii escirleiiririii (cv. Moneymaker)

Good to moderate SOk7lZUll2 ll1~1111170S11171

Trifoliuiiz repens') I .. Poor -

Achillea iizillefoliriii Solailuin izigruiu

~ Non Coizyza canadeilsis Senecio clivicola Urtica dioica

Resistant L. esculeiztunz (cv. Rossol)

LSD (P = 5%)

27.0 35.8

26.6 -1 )

16.0 16.0

11.8 8.2 3.8 12.6

0.0 0.0 3.6 - 0.0 1.6

2.6' -

4.44

34.2

29.6 1 9.2

2.4 7.8

0.0 0.4 0.0

12.6

33.0 30.6

- 32.4 15.6 17.4

0.8 4.2 11.0 3.6

3.6

-

3.4

0.4 6.0

0.0

0.0 -

- 2.80

0.0 0.0 0.0

0.0

0.0

0.0 -

-

2.41

') (-) not tested with L15. ') Final harvest was after 23 days.

nita eggs (O, 2000 and 40 000/eggs per pot, equivalent to O, I .O and 20.0 eggs/g soil) were applied to give 1 O replicates of each plant specieshematode population density. The inoculum comprised an equal mixture of NaOCl extracted eggs from populationsL15, L19 and L20. Aldicarb (Temik 10G) was applied and mixed into further groups of five pots with O or 40 O00 eggs/pot to be planted with either tomato cv. Moneymaker or U. dioica. Two days later each pot was planted with two seedlings of the test plants, spaced so that one seedling could be removed 15 days later without damaging the other. The pots were maintained in a glasshouse at about 25°C during the day and about 22°C at night.

At planting the water content of the soil in the pots was near to field capacity. After 3 days, all pots were watered daily as necessary. The pots were randomized within 10 blocks which were rotated and re-randomized each week. Fertilizer was added weekly as a liquid feed (Vitafeed III) and the plants were sprayed twice with bupirimate and n- butanol (Nimrod) to control downy mildew on the tomato

426

and with fenbutantin oxide (Torqua) to control red spider mite on U. dioca.

To determine initial rates of invasion, one seedling with all its roots was dug out after 15 days, by which time all the eggs in the inoculum should have hatched. After 45 days, the roots of five replicates for each treatment (but not the aldicarb treated) were washed free of soil in a bucket. The numbers of galls, plant height, total fresh root (blotted dry) and shoot weights, and shoot and root dry weights (80°C for 2 days) were determined. The number and stages of development of the nematodes were also determined in chopped and mixed, 2 g root sub- samples stained with acid fuchsin. The remaining five replicates were harvested after 88 days and the same data as at the first harvest were recorded. To determine the number and stages of development of the nematodes, the root system of each plant was chopped into short lengths, mixed, and a 10% sub-sample stained with acid fuchsin.

Nematology

Meloidogyne host statzis aiid damage

Table 5. Percentage of the popiilatioiz wlziclz were iiiideveloped secoizd stage juveiiiles (J2), developed J2 aiid yowig feiiiales at 15 days after iiioculatioii.

Population L20 Population LI5 Host status % Undev. % Dev. % Young % Undev. 9 Dev. % Young

Plant species * J2 J2 fernales J2 52 females

- Good Lycopersicoiz esciileiitiiiii cv. Moneymaker

Good to moderate Solaiilciii ~ i ~ a i i i ~ i i o ~ ~ i ~ i i

Trifoliuiiz repens

Poor Achillea iiiillifoliuriz

. Solailuin izigruiii

10 47 42

O

O 39 60

30 46

70 52

- 86

- 1 2

67 18

' 33 56

O O O

O 26

50 6

50 76

O 18

Non _.

Coizyza calmdensis fenecio clivicola ,iJrtica dioica

'L. esculeiztuin cv. Rosso1

I Rhistant

'. o 100 O

O O O

O

O -

O

O -

O

O -

100 O O

I)-, not tested with L15.

i DATA ~ N A L Y s I s Corzyza caizadeizsis, Urtica dioicn (Table 11, and Seiiecio

P clivi.cola (Table 2). Egg masses were observed on the

/zihpidus (from the UK), Daturaferos (Botswana), and irzrzoxia (Namibia).

'1

There were significant differences in gall production ( P . < 0.05) between populations; population L27 (in- ocufum only 300 eggs per pot in Experiment 2) induced fewest galls with many of the individual species of plants, and overall, in both experiments: Galls were produced by at least two of the four populations of M . incognita on 22 of the 24 mainly wild, temperate plant species (not including tomato) tested in Experiment 1 (Table l), and 32 of the 33 of the species/accessions, mainly from warm climates, in Experiment 2 (Table 2). Of 57 species/accessions tested in both experiments, 50 were galled by all four popula-

Species of plants differed in their susceptibility to the $ffg~ nt populations. The ANOVA showed that there was a significant influence ( P < 0.001) on mean gall numbers of nematode Population and Plant species. However, as mean root system weight ranged from 0.3 g for E. vagi-

The package, (Payne et 'l.2 1997) roots of all plants which produced galls except Leoiztodolz was usid for all analyses and least significant differences (LSD, k < 5%) are presented. However, as all experiments ibvolved the analysis of results from different plant species! which often grew to different sizes, the utility of some c,&mparisons is partly a matter of judgement. Gener- ally, anLSD will provide an under, rather than an over es- timat$of differences. Roodplant weights were measured and $e given to inform and facilitate judgements.

iT ,q ,$

Results li? 1' .,. .

.ASSESSMENT OF HOST STATUS TO FOUR POPULATIONS OF M. INCOGNITA

Experinzents 1 and 2

On susceptible tomato cv. Moneymaker all fourpopula- tions produced 50 to 100 galls and numerous egg masses in.both experiments (Tables 1 and 2). No galls or egg masses 'were produced by any of the four populations on

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Vol.. .1(4), 1999 427

M. E. Eliwaeti et al.

Table 6. Percentage of the yopihtioii at direreizt stages of development and rzunzber of egg inasses (and eggsleg8 inass) at 35 days after iiioculatioii (iiieaiis of jve replicates).

Population L20 Population L15 Host status 9 Undev. %'Dev. % NO % Undev. % Dev. % No

Plant species . J2 J2 females egg mass 52 52 females egg mass

Good - Lycopersicoiz esciileiiruiii O O 100 10.2 (241) O O 100 14.0 (276) cv. Moneymaker

Good to moderate 84 5.4 (66) -3) - - - - Solarzuin ~~zariziiios~iii~ O 16

Trifolium repens2) 35 55 10 O 5 94 O O

Achillea inillifoliiiiii 24 24 52 1.4 (90) O 1 O0 O O Poor -

O O 100 4.2 (288) Solaiiuiii iiigruiii O O 1 O0 2.0 (71)

- Non Coiiyza canadeizsis O O O O O O O O Senecio clivicola O O O O Urtica dioica O O O O O O O O

L. esculentunt O O O O

- - - -

Resistant - - - - cv. Rosso1

Mean number of eggslegg mass; LSD ( P < 5%) = 70. Harvested after '23 days.

3)-, not tested with L15.

naturn to 13.5 g for C. unizuin (Table 1) this may have sometimes influenced gall numbers. Even so, E. vagina- turn (cotton grass) produced more galls overall than C. aiziziun. There was also a significant ( P 6 0.001) interac-

example, S. nigruni was a much better host, and Epilo-

than an accession of the same species from Africa (Table 2). However, the unknown accession produced a mean root weight of 1.4 g compared with 0.5 g for that from Africa.

EFFECTS ON INVASION AND DEVELOPMENT tion between plant species and nematode population. For 1

biirriz lzirsutum was a much poorer host for L15 than for the other three populations (Table 1).

There was no obvious trend for a population from one part of the world to produce most galls on plants from the same region, except for L19 from Martinique which consistently induced a significantly ( P 6 0.05) greater number of galls than the other populations on four of the six Nicotiana spp. (Table 2). Two accessions of Chenopodium album and of C. inurale were tested in

Experiment 3 Five days after inoculation, the plants selected as mod-

erate to good hosts (S. niaiiznzosuiiz, T. repens, L. esculen- tuin cv. Moneymaker) generally had a greater total root length, more root tips and more juveniles (52) per root tip than those selected as poor hosts (A. rnillefoliunz, S. izigrum). These, in turn, had longer root systems than the non-hosts (S. clivicola, U. dioica, C. canaderisis) (Ta- ble 3).

Experiment 2 and C. album from North America was a significantly ( P 6 0.05) better host for populations L19 and L20 than the second accession from N. America. Conversely, C. murale of unknown origin was a better host for all populations, and particularly for L15 and L19,

The good hosts were consistently and significantly ( p 6 0.05) the most heavily infested (from 16 to 31 J2/plant) and the mean numbers per plant changed little between 5 and 35 days after inoculation (Table 4). How- ever, I: repens contained consistently fewer nematodes

328 Nematology

. . . . - . . . , *

..:,~:; , . ....,.. .. . . . " . I .

. _ _. . , . ,,. .-:. ,

_ . . . . . .

/

Meloidogyne host sratrrs arid daiiiage

than S. iizai?zinosuiiz and tomato cv. Moneymaker. The two poor hosts (S. izigruiiz, A. iiiillefoliuiiz) contained fewer nematodes than the good hosts and numbers tended to de- crease between harvests (a mean of 9.1 /plant at 5 days, 5.5 at 15 days and 3.6 /plant at 35 days). S. izigniiiz infested with L15, for which it is a good host, contained significantly ( P 6 0.05) more nematodes than A. mille- f o h n (Table 4). Of the three non-hosts, C. caizndeizsis contained no J2 at any harvest, U. dioica contained a few 52 of L20 only at the day 5 harvest, and S. clivicola contained a few 52 only at the day 5 and 15 harvests. Resistant tomato cv. Rossol contained 52 at days 5 and 15 but none at day 35.

At day 15, in tomato cv. Moneymaker40% and 60% of L20 and L15 respectively were young females (Table 5). In T repens only 2 and 1 % had developed into ypung females and in S. i~iaiiziizosuin none had developed into females. In the poor host S. izigruiiz, 26% and 18% were female but none were observed in A. inill$oliunz. In resistant tomato cv. Rossol, all juveniles were vermiform and undeveloped.

After 35 days, egg masses were present on S. izigrunz, S. niaiiziizosuiiz, tomato cv. Moneymaker, and on A. milli- foliuiiz inoculated with L20 (Table 6). The number of eggs per egg mass was greatest on S. izigruiiz infected with L15, closely followed by tomato cv. Moneymaker. Population L20 had produced some females on T repens when it was harvested at day 23.

Only small numbers of juveniles were extracted from the soil 5 days after inoculation (results not shown). Fewest $ame from the pots growing susceptible tomato cv. Moneymaker (a mean of 1.6 J2/pot) and most from resistant tomato cv. Rossol (8.8 J2/pot). At day 15 juveniles were recovered only from pots growing U. dioica, S. rnaiiuizosuiii, and susceptible and resistant tomato (means of 9.0,5.8, 1.0, and 8.8 J2lpot respectively).

e .

HOST STATUS AND DAMAGE

Experiment 4 Rates of invasion 15 days after planting (Table 7) were

as expected from the previous experiments. Susceptible tomato cv. Moneymaker was the most heavily infested, S. izigruin and D. iizetel contained substantially fewer J ~ s ,

Table 7. Root weight aiid nuiirbers of iteiiiatodes i12 foirr species of plants 15 days after inoculatioir with n ~ o derisiries of Meloidogyne incognita (means of 5 replicates).

Host status Pi Root No of

soil plant Plant species eggslg - wt(g) nematodes/

¿

Good Lycopersicon esculeizfioiz (cv. Moneymaker)

Intermediate to poor Datura inetel

Non Urticn dioica

LSD (P < 5%)

1.0 0.36 20.0 0.38

1.0 0.10 20.0 0.16

1.0 0.16 20.0 0.14

1.0 0.20 20.0 0.22

0.08

5.0 83.0

0.8 1.6 2.6 9.6

0.0 0.0

13.1

After 88 days, compared with the uninoculated plants, tomato height and shoot fresh weight (and total fresh and dry weights - data not shown) were significantl? less ( P < 0.05 or GO.01) for plants infested with the highest inoculum density (Table 8). Root fresh (and dry) weights were greater ( P < 0.05) than the control. The growth of the other three species of plants had not been significantly affected. Aldicarb (Temik 10G) (Table 8, results in brackets) did not affect the growth of the uninoculated control plants but it increased significantly ( P 6 0.05) the shoot growth of the tomato with the highest inoculum density (Pj 7 20 eggs/g soil) and prevented the increase in root weight associated with galling.

Numbers of galls on day 45, and nematodes in the roots and eggs 45 and 88 days after planting (Table 9) confirmed that U. dioica is a non-host and D. iizetel a poor host for the M . iizcogizita population mixture. On day 45, females and eggs were present in S. izigriiin and L. escirleirtuiii but only undeveloped and developed juveniles were found with D. iizetel. On day 88, some eggs were found in D.

and U. dioica none.

only the growth of tomato cv. Moneymaker had been affected, and then only by the highest pí (20 eggs/g soil). The growth of the other three species had been unaffected (Table 8).

.',. metel, but the population had decreased ( P f / P f = 0.4

After 45 days, compared with the uninfested plants, ".. and 0.12 respectively with Pi = 1.0 and 20 eggstg soil). .''The population had increased on S. izigr~11?z ( P f / P i = 2.6.:@ 2.5 respectively) but it had increased most on susceptible tomato cv. Moneymaker ( P f / Pi = 12.2 and 1 O .2 respectively).

Vol. 1(4), 1999 429

M.E. Elzwaeti et al.

Table S. Effects of Meloidogyne incognita 011 the growtlz offour plaiit species 45 arid 88 days after iriocirlatiori (iiiearis of 5 replicates).

45 days 88 days

Host status Pi Plant Shoot Root Plant Shoot Root Plant species (eggs/g height fresh fresh height fresh fresh

soil) weight weight weight weight (cm) (g) (d (cm) (g) (8)

Good Lycopersicorz 0.0 65 84 6.2 134 (136)') 234 (246) 18.0 (15.0) esculenturit 1 .o 63 72 5.6 135 230 20.6 (cv. Moneymaker) 20.0 52* 65" 8.6 109* (129) 120* (282) 33.4* (19.0)

Intermediate to poor Datura inetel 0.0 35 51 9.0 70 128 28.2

1 .o 34 51 9.4 94 140 30.8 20.0 45 50 8.2 83') 121 24.2

Solarzuin riigrurn 0.0 74 59 10.2 99 139 12.2 1 .o 70 51 9.4 102 129 10.2

20.0 83 67 13.0 106 13 1 19.0

Non Urtica dioica 0.0 64 32 6.6 120 (145) 73 (77) 23.6 (28.6)

20.0 63 29 5.8 135 (135) 73 (84) 26.0 (16.8) 1.0 65 41 8.4 134 65 18.4

LSD (2' = 5%) 13 13 2.9 20 (30) 32 (48) 8.9 (6.3)

'1 Results in brackets are for plants in soil treated with aldicarb at planting. 2, The LSD is based on results for all plant species. It will, therefore, tend to underestimate differences within species. .,. Significantly different from uninoculated plants P < 5%.

Discussion

Host status appears to vary because of differences in rates of invasion and/or development. Most poor hosts appear to be less heavily invaded than good hosts (Table 4), and the mechanisms involved in deterring/preventing invasion must be worthy of further analysis. In some hosts where invasion was decreased, the rate of juvenile development, and fecundity were also decreased (e.g. Acliillea inillefoliunz), but in others (e.g. Solanum izigruiiz with L20) the rate of development was almost equal to that in susceptible tomato (Table 5). Mortality, and/or the proportion becoming male were also increased in some poorer hosts.

The hypothesis that most wild, herbaceous plants from all parts of the world are potential hosts for M. iizcogizita was supported by the results of Experiments 1 and 2, in which even a plant such as Eriophoruin vaginatuin, from marshy, mountain areas in the UK, was a host. These results also support the suggestion that the mitotic partheno-

genetic Meloìdogyrze spp. have evolved, and conserved genomes which make most plants susceptible (Trudgill, 1997). Overall, at least two of the four populations of M. incognita induced galls on all excegt three of the 57 species of plants tested and egg masses were observed on 5 1 of 57 species.

The converse hypothesis, that resistance has not neces- sarily resulted from CO-evolution between pathogen and plant, was also supported. Some species appeared to be resistant tohon-hosts for one or more populations of M. incognita (e.g. Capsicuin anmuin, Solarzuiiz sodaizaeunz, Leoiztodoii taraxacoides - from tropical America, from Africa and from the UK respectively, Tables 1 and 2) but hosts for the others. Coizyza caiiadeizsis, an introduction into the UK from North America, was a non-host for all populations and appeared not to be invaded. Urtica dioica and Senecio clivicola, from the UK and South America respectively, were non/resistant hosts, but they were invaded, albeit by only small numbers of J2.

430 Nematology

Meloidogyne host status and darnage

Table 9. Nuilibers of galls 45 days aftel- iizoculation aiid of Meloidogyne incognita (niixtiire of yopiilntioris LlS, L19, L20) 35 aiid 88 days afer iizocidatioii (ineatis of 5 re,vlicares).

~-

45 days 88 days Host status Pi No No No No Total NO No Total Reproductive

Plant species (eggdgsoil) galls J2s & eggs nematodes nematodes eggs rate ( P f / P i ) I

Good Lycopersicon 0.0 O 0 o O O O O O -

(cv. Moneymaker) 20.0 1944 665 3690 33220 37575 122590 199700 408600 10.22

Datura ilzefel 0.0 O 0 o O O O O O -

esciilei~t~iiii 1 .o 91 O 170 1330 1500 11300 13000 24300 12. I5

Intermediate to poor

1 .o 4 45 o O 45 500 300 800 0.40 20.0 231 690 O O 690 1600 3300 4900 0.12

1 .o 44 O 220 485 705 2900 2300 5200 2.60 20.0 930 80 2235 18660 20935 36700 61700 98400 2.46

Solariuni iiigriuii 0.0 O 0 o O O O O O -

Non - Urtica dioica 0.0 O 0 o O O O O O -

1 .o O 0 o O O O O O O 20.0 O 0 o O O O O O O

LSD (P = 5%) 117 326 740 5921 6565 33092 42912 51376 -

The majority of plants tested were poor (t10 galls/ plant) or non-hosts for one or more populations. Some were relatively good hosts (>50 galls/plant) for one population, but a poor host for the others e.g. S. nigrunz. Such a strong plant x nematode interaction indicates a "gene for gené'', resistancelvirulence interaction. However, the effects on juvenile invasion and development of the resistance in tomato cv. Rossol, conferred by the major gene, Mi, appeared to be different to those observed in the poor host status studied here. The Mi gene slowed the invasion of the J2, but substantial numbers accumulated over 15 days. Yet, after 35 days, none were found in cv. Rossol, because either they had died, or had left the roots due to the rapid hypersensitive response to invading, avirulent 52 (Paulson & Webster, 1972). No galls were formed on CV. Rossol, indicating that none of the 52 had established a feeding site. In contrast, although fewer J2 invaded most of the poor hosts than invaded susceptible tomato cv. Moneymaker, they did induce galls and some.. eventually developed into egg-laying females. However, ''k... as observed by Anwar et al. (1994), rates of development and fecundity were often less on poor than good hosts. Consequently, in some poor hosts the Same mechanism might be involved in decreasing both invasion and de-

velopment, suggesting a possible parallel with the races of Dityleitclzus dipsaci and the differences in their host ranges.

Differences in host status were, as hypothesised, associated with differences in the tolerance of damage by M. -incognita. Only susceptible tomato (L. escrrleitruiiz cv. Moneymaker) was damaged by the highest inoculum density, Pi = 20 eggs/g soil. Even S. r~igruiiz, which was a moderate host because the mixed inoculum contained some LI5 (33% each of LIS, LI9 and L20), was not damaged. Two factors were probably involved; fewer 52 initially invadedhecame established, and the population increased more slowly than on fully susceptible tomato. Ehwaeti et al. (1998b) showed that, in a long-term experiment in large pots, the population density of M. iiicogriita increased so greatly over several generations that damage to tomato cv. Moneymaker intensified as the growing sea- sonprogressed.

Host status affects not only the damage a plant is likely to suffer when planted in soil infested with M. iitcognira,

-. but also the population left at harvest to attack the next c&$@I the rotation. some weeds are, therefore, likely to be more important than others as alternative hosts, sup- porting the observations of Madulu and Trudgill (] 993).

Vol. 1(4), 1999 43 1

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M.E. Elwaeti et al.

Although the research here studied mainly wild plants, the same principles almost certainly apply to crop plants. The variation in plants as hosts for different populations complicates the use of poor hosts in the management of M. irzcog~zita. However, legumes which are poor hosts for most populations e,g, Crotalaria spp. may be useful in rotations designed to both increase fertility and suppress Meloidogyrze spp. (Madulu et al., 1994).

.

Acknowledgements

Drs K. Thompson (University of Sheffield) and S. Lin- ington (Royal Botanic Gardens, Kew) for supplying the seeds of most of the plants used, Mrs A. Holt for main- taining so carefully the populations of M. incognita tested. The first author was supported by the government of Libya, and the Scottish Office Agriculture, Environment and Fisheries Department financed the facilities used. The work was part of an international collaborative project TS3*' CT92-0098 funded by the European Union.

References

ANWAR, S.A., TRUDGILL, D.L. & PHILLIPS, M.S. (1994). The contribution of variation in invasion and development rates of Meloidogyrie iiicogiiita to host status differences. Nerrzatologica 40,579-586.

BIRD, A.F. (1972). Quantitative studies on the growth of syn- cytia induced in plants by root knot nematodes. biteriiatioiial Josrriial of Parasitology 2, 157-170.

BLOK, V.C., EHWAETI, M.E., FARGETTE, M., KUMAR, A., PHILLIPS, M.S., ROBERTSON, W.M. & TRUDGILL, D.L. (1997). Evolution of resistance and virulence in relation to the management of nematodes with different biologies, origins and reproductive strategies. Neiiiatologica 43, 1- 13.

EHWAETI, M.E., PHILLIPS, M.S. & TRUDGILL, D.L. (1998). The viability of Meloidogyrie iricogrzita eggs released from egg masses of different ages using different concentrations of sodium hypochlorite. Neiizatologica 44, 207-2 17.

EHWAETI, M.E., PHILLIPS, M.S. & TRUDGILL, D.L. (1998). Dynamics of damage to tomato by Meloidogyiie iiicogiiita. Fuiidariieiital aiid Applied Neriiatology 21, 627-635.

FERRIS, H., SCHNEIDER, S.M. & SEMENOFF, M.C. (1984). Distributed egg production functions for Meloidogyrie nre- riaria in grape varieties and consideration of the mechanistic relationship between plant and parasite. Jouriial of Neriiatol-

HOLBROOK, C.C., KNAUFT, D.A. & DICKSON, D.W. (1983). A technique for screening peanuts for resistance to Meloido- gyrie arerieria. PIarit Disease 67, 957-958.

HUANG, S .P. (1986). Penetration, development, reproduction, and sex ratio of Meloidogyrie javanica in three carrot cultivars. Jo~rrrial of Neriiatology 18,408-41 2.

HUSSEY, R.S. & BARKER, K.R. (1973). A comparison of methods of collecting inocula of Meloidogyiie spp., including a new technique. Plant Disease Reporter 57, 1025-1028.

JEPSON, S.B. (1987). Ideiitijïcatiori of root-knot rieriinrodes (Meloidogyne species) Wallingford, UK, CAB International, 265 pp.

MADULU, J.D. & TRUDGILL, D.L. (1993). Weed hosts of Meloidogyiie javariica in Tanzanian tobacco fields. Pakistan Joui-na1 of Nerizatology 11, 61-64.

MADULU, J.D., TRUDGILL, D.L. & PHILLIPS, M.S. (1994). Rotational management of Meloidogyne javaizica and effects on Pasteuria penetraris and tomato and tobacco yields. Ne- iizatologica 40,438-455.

PAULSON, R.E. & WEBSTER, J.M. (1972). Ultrastructure of hypersensitive reaction of roots of tomato Lycopersicori escu- lentuiii L., to infection by the root-knot nematode, Meloido- gyne iizcogiiita. Pliysiological Plaiit Patliology 2,277-234.

TRUDGILL, D.L. (1997). Parthenogenetic root-knot nematodes (Meloidogyrie spp.); how can these biotrdphic endoparasites have such an enormous host range? Plaiit Patlzology 46, 26- 32.

O ~ Y 16, 178-183.

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