Journal of Chemical Ecology, Vol. 15, No. 2, 1989
CHARACTERIZATION A N D PARTIAL PURIFICATION OF ATTRACTANTS FOR NEMATODE Orrina phyllobia FROM FOLIAGE OF Solanum elaeagnifolium
A.F. ROBINSON
A N D G. S A L D A N A
Agricultural Research Service, USDA Subtropical Agricultural Research Laboratory Weslaco, Texas 78596 (Received November 9, 1987; accepted January 8, 1988) Abstract--An unknown attractant for the nematode Orrina phyllobia was extracted with water from foliage of Solanum elaeagnifolium. Stability, solubility, ionic character, and chromatographic purification were investigated using a bioassay based on nematode aggregation in agar. Activity was nonvolatile, dialyzable, heat stable below 60~ and partially lost within 30 min at 100~ Activity was unchanged from pH 5 to 12, but was entirely lost at pH 2. Loss of activity at low pH did not appear to result from direct effects of pH on nematode behavior and was partially recovered by readjustment to pH 7. The attractive factor was most soluble in water and appeared to be cationically but not anionically exchangeable. Activity appeared to chromatograph as several compounds by high-performance liquid chromatography employing reverse phase C~8 and amine-bonded columns. Various known compounds that are common to Solanum spp. or that attract other nematodes were unattractive. Extraction of S. elaeagnifolium foliage specifically for solanaceous glycoalkaloids using methods developed for S. tuberosum did not yield an attractive product. Key Words--Orrina phyllobia, Solanum elaeagnifolium, foliar nematode, host finding, nematode chemotaxis, silverleaf nightshade.
INTRODUCTION
M o r e than 4 0 0 0 n e m a t o d e species parasitize plants and considerable research has b e e n d o n e t o w a r d learning h o w s o i l - b o r n e i n f e c t i v e stages find roots and stems ( r e v i e w e d by Steiner, 1925; Croll, 1970; G r e e n , 1971; Prot, 1980; Z u c k e r m a n and J a n s s o n , 1984). N e m a t o d e orientation to t e m p e r a t u r e and soil m o i s 481
482
ROBINSON AND SALDANA
ture gradients may assist host finding, but most evidence indicates chemotaxis is primarily responsible. Very little is known concerning specifically phytogenic attractants. Many nematodes are attracted to root leachates; in at least two cases, root leachate attractiveness was removed with activated charcoal, suggesting an organic nature (Miller and Mclntyre, 1976; Peacock, 1961). Various common biogenic chemicals, largely amino acids, attract or repel some bacteriophagous, phytoparasitic, and zooparasitic nematodes on agar or agarose gels (Dusenbery, 1983; Pye and Burman, 1981; J.B. Ward et al., 1984; S. Ward, 1973). These include L-tryptophan, glutamic acid, aspartic acid, cysteine, L-tyrosine and other amines, cAMP, cGMP, gibberellic acid, glutathione, ascorbic acid, and pyridine. These responses were discovered by screening hundreds of known compounds and none has been established to mediate orientation within soil (Dusenbery, 1983). Failure to identify plant-specific attractants has encouraged the view (Klingler, 1965; Prot, 1980; Wallace, 1973) that phytoparasitic nematodes in soil may rely primarily on nonspecific chemical stimuli, such as CO2 and inorganic ions for orientation. Several species orient on gradients of CO2, Na +, CI-, or O H - . Progress toward understanding nematode chemotaxis as it relates to specific plant hosts has been made with the pine-wood nematode, Bursaphelenchus xylophilus (Futai, 1980). Various terpenes and other compounds common to the coniferous plants are attractive or repellant in vitro (Tominaga et al., 1983, 1984). The life cycle of this nematode differs from most phytoparasitic species in that it is normally transmitted to the trunk and branches of the host tree by a pine sawyer beetle (Monochamus alternatus), rather than by moving through soil. Thereafter, nematodes move and feed within resin canals where behavioral responses to terpenes may play an important role. Chemotactic preferences for tree saps appear related to host suitability among tree genera but not among pine species (Futai, 1980). A second nematode whose behavior appears attuned to specific plants is Orrina phyllobia. It is known to parasitize only foliar parts of certain plants within the genus Solanum and is specifically attracted to leachates from stems of four Solanum spp. These are S. tuberosum (potato), S. melongena (eggplant), S. carolinense (Carolina horse nettle), and S. elaeagnifolium (silverleaf nightshade). Leachates from rhizomes of Solanum spp. and from stems of 30 plant species in other genera spanning 12 plant families were not attractive (Robinson et al., 1979). Attraction to stems is ecologically valuable to O. phyllobia because its life cycle entails aggregation of infective juveniles, which are about 700 tzm long, within soil around stem bases and emerging shoot tips. When stem surfaces are moist, the juveniles ascend as far as 50 cm and invade foliar meristems to initiate new infections (Robinson et al., 1978). We have begun to characterize and chromatographically purify attractant from foliage of the common host,
483
NEMATODE ATTRACTANTS
S. elaeagnifolium, with a view toward understanding this rather host-specific behavior by a phytoparasitic nematode.
METHODS AND MATERIALS
Bioassay Procedure. Stems and leaves of S. elaeagnifolium were collected from field-grown plants, freeze dried intact, and ground with a Wiley mill. The powder obtained was stored in a desiccator over anhydrous CaSO 4 and used as a standard source of attractant. Preparations from plants collected on other occasions were similarly attractive. The assay we adopted was a modification of methods described by Balan et al. (1976) and Tominaga et al. (1983) for studying chemotaxis in other nematodes. Infective juveniles of O. phyllobia were removed daily from dried leaf galls of S. elaeagnifolium as previously described (Robinson et al., 1979) and suspended in a continuously aerated dilute salt solution (4.5 mM NaC1, 0.4 mM KC1, 0.05 mM CaC12, 0.05 mM MgC12) to reduce ion regulation stress. Bacto-Agar (1.5%, Difco) was dissolved in water (w/w) supplemented with dilute salts equivalent to those in the nematode suspension. Equal parts of nematode suspension (22 ~ and agar (42 ~C) were then mixed and poured into a 35-ram-diameter Petri dish to obtain a 2-ram-deep film of 0.75 % agar containing about 3000 juveniles. The mixing procedure induced a brief reduction in motility from which nematodes completely recovered within several minutes. A 3-mm-diameter disk of filter paper (Whatman No. 1) saturated with the test solution and air-dried was placed halfway between the center and the edge of the dish. Trials with aqueous solutions of the biological stain safranin-O indicated that the pattern of diffusion of material from the dried disk was hemispherical downward. Two untreated disks were then placed equidistant from the center, 1 cm from each side of the treated disk. After 90 min, a 5-ram cork borer and small spatula were used to transfer agar cylinders containing the disks to counting dishes where the nematodes within each cylinder were dispersed into water and microscopically counted. The response index (RI) was defined as RI = T/(T + U) where T = nematodes under the treated disk and U = average number of nematodes under untreated disks in the same dish. RI > 50 % indicated aggregation and RI < 50 %, repulsion. RI values were binomially distributed and examined statistically after arcsine transformation. In each experiment except where noted, five dilutions spanning two orders of magnitude were prepared for bioassay from filtered aqueous extract of the dry plant powder (6 % w/w). The 6 % concentration will be referred to as standard foliar extract (SFE). Nematode chemotaxis experiments are frequently conducted without light to mimic soil conditions. In preliminary experiments, we found that bioassay
484
ROBINSON AND SALDANA
results obtained under laboratory light were identical to results obtained in the dark. Small temperature gradients (ca. 0.1 ~ that resulted from differential evaporation of water within dishes strongly influenced nematode distributions. However, dishes were covered and oriented identically to minimize the effect. We recognized that test solutions might generate behaviorally important extremes of pH, ionic strength, and osmotic potential. To evaluate their contributions, we bioassayed aqueous solutions of acetic acid, HC1, and NaOH at 0.01, 0.1, 1, 10, and 100 raM. We also bioassayed solutions with osmotic pressures from 100 to 500 mM by supplementing water and foliar extract with sucrose or inorganic ions in ratios described. Standard foliar extract had 100120 mM osmotic pressure by freezing point depression and pH 6.3-6.5. Bioassays were performed in five replicates. Experiments were repeated at least once. Reproducibility was sometimes confirmed by assigning numerical ratings to aggregations instead of counting nematodes. Preliminary Characterization of Attractants. Standard foliar extract was bioassayed after each of the following procedures: dialysis against distilled water across 3500 MWCO dialysis tubing; filtration through activated charcoal; heating to 50, 60, 70, 90, and 100~ for various intervals up to 75 min; pH adjustment with HC1/NaOH; and readjustment to pH 7. Extract had appreciable buffering capacity near pH 6.5, and neutralization beyond 3 pH units required excessive dilution of extract or increases in Na § and C1- concentrations > 200 raM. These are physiologically significant levels; therefore, control solutions were prepared with similar osmotic pressures by adding NaC1 to extract without pH adjustment. To examine attractant solubility, foliar powder was extracted (2 % w/w) with solvent mixtures in a polarity series including water, methanol, 95% ethanol, 1-propanol, 1-butanol, diethyl ether, and hexane. Each extract was then filtered, evaporated to dryness, and reextracted with water at original volume. Residue obtained from the first filtration was separately reextracted with water and all solutions assayed. Ionic character was examined with prefilled Poly-Prep columns (Bio-Rad) containing 2 ml of AG 50W-X8 and AG l-X8 ion exchange resins in the H + and C1- forms. Aqueous extract was prepared at 67% the concentration of SFE. After washing with distilled water, resin was drained under vacuum, and 3 ml of extract was passed through each column. Columns were then washed with 9 rnl distilled water and eluted with 4 ml 100 rnM KNO3, followed by another 4 ml 1 M KNO3. Eluates and washes were bioassayed before and after adjustment to pH 7 with NaOH. Toward HPLC Purification. Preliminary separations were made with a Waters HPLC system equipped with a reverse phase Cls Semi-Prep column (7.8 m m x 30 cm) (Waters) and a variable wavelength detector set at 254 nm. Solvent flow was 1.5 ml/min as follows: 15 rain isocratic water, 10 min linear gradient to 100% methanol, 10 rain hold, 20 rain linear gradient to 100% water. Eleven 5-rain fractions were collected separately from three 50-/zl injections of
NEMATODE ATTRACTANTS
485
67% SFE, composited, evaporated to dryness (60~ redissolved with 2 ml water, then reevaporated to dryness in small dishes under vacuum (50~ and finally redissolved in 150 #1 water for bioassay. Solvent controls were also tested. A cleanup procedure using disposable C~8 preparatory columns (Sep Pak, Waters) was then developed based on the column volume equivalents of eluants needed for a stepwise approximation to the HPLC solvent gradient. Active fractions were prepared in quantity with the cleanup procedure. Further separations were done with an analytical C18 Radial Pak cartridge (8 mm x 10 cm) or an amine-bonded column (3.9 mm x 30 cm) (Waters). Mobile phase for the C18 cartridge was a 25-min water-methanol linear gradient ( 1.0 ml/min) from 20 % to 80 % methanol. Mobile phase for the amine-bonded column was a 20-min CH3CN-water linear gradient (2.0 ml/min) from 90% to 65% CH3CN, a 5-min hold, and a linear gradient back to inititial conditions. Thirtyfive 1-min fractions were collected separately from two injections, composited, evaporated, reextracted with 25 ~1 water, and bioassayed. Examination of Known Substances for Activity. Commercial preparations (Sigma) of various compounds were bioassayed because they are associated with Solanum spp. or because they have chemotactic activity to other nematodes. These included the common solanaceous glycoalkaloids c~-chaconine and o~-solanine at pH 7.5. With the view that attractants might occur among other Solanum-specific glycoalkaloids, the procedures described by Allen and Kuc (1968), by Bushway et al. (1985), and by Carman et al. (1986) for extracting glycoalkaloids from potato were applied to the S. elaeagnifolium foliar powder and confirmed by HPLC of product against commercial preparations of o~-chaconine and a-solanine.
RESULTS AND DISCUSSION
Bioassay Procedure. The RI reached equilibrium after ca. 60 min (Figure 1A). The RI always approximated a log decay function of concentration that asymptotically approached 85-95 %. The least-squares line relating R1 and log10 concentration was employed to express bioassay results in terms of the apparent concentration of attractant as well as in RI units (Figure 1B). Results of one experiment (Figure 3 below) are presented in both ways to convey the exponential relationship between attractant concentration and statistical uncertainty. Independent effects of pH on the bioassay were not detected with acetic acid and HC1 solutions of pH > 2. NaOH > 10 mM caused weak aggregation and at pH 12 was equivalent to the effect of 3% SFE. Adding sucrose or mixed salts to water and foliar extract to generate osmotic pressures as high as 500 mM did not change bioassay results. Osmotic pressures of test solutions in subsequent experiments were < 300 mM. Some species of nematodes respond
486
ROBINSON AND SALDANA i
A 7s
z REPULSION
25
RESPONSE TO STANDARD FOLIAR EXTRACT
2'o
,'o
2~
8'~
DURATION OF ASSAY (MINUTES1 101~
B
i
i
J
t
~ 6a
CONCENTRATION (kOGlo STANDARD FOLIAR EXTRACT)
FIo. 1. Effects of bioassay duration and foliar attractant concentration on the nematode response index. (A) Increase in nematode response during 90-min exposure to aqueous extract of Solanum elaeagnifolium foliage. (B) Representative concentration-response curve for aqueous foliar extract. Concentrations are expressed as percentage of a standard concentration of dry foliage in water (6 % w/w). Brackets indicate confidence intervals for five replicates obtained after arcsine transformation. The straight line and the sigmoid log decay function [dy/dx = (Y - y)K, where y = response index, x = concentration, Y = the maximum value of y, and K = a constant] were fitted by least squares.
behaviorally to pH, ion, and osmotic pressure gradients much smaller than we imposed (Dusenbery, 1983; Prot, 1980). I f O. phyllobia also responds to them, their effects are very small under our assay conditions compared with the effects o f attractant within S. elaeagnifolium foliage. Preliminary Characterization of Attractants. Attractant from S. elaeagnifolium was essentially nonvolatile, freely dialyzable, and heat stable for 90 min under the conditions we u s e d for evaporating test solutions ( < 6 0 ~ How-
NEMATODE
487
ATTRACTANTS
ever, some activity was lost within 30 min at 100~ (Figure 2A). Further losses were not detected up to 90 rain, suggesting that heating generated a repulsive product or that two or more attractants were present and differed in temperature sensitivity. Activity was highly polar or ionic in character, not removed with activated charcoal, and most soluble in water (Figure 3). The attractiveness of standard fotiar extract was completely lost by adjusting pH to 2 with HC1 but was partially recovered after readjustment to 7 (Figure 2B). Addition of NaC1 to standard foliar extract at concentrations equal to those generated by pH adjustment did not reduce activity, suggesting that direct effects of Na + and C1- on nematode behavior were not important factors. Disks with SFE at low pH frequently induced nematodes to aggregate in a halolike ring surrounding the disk 2-4 mm from its edge. When a second disk, which had been saturated with 0.1 N NaOH and dried, was placed on top of the first disk, the nematodes J
,
,
A
ATTRACTION ................................................ REPULSION
60 i00
TINE
THAT
J
EXTRRCT
WAS
i
HEATED
(NINUTES)
i
J
C
•
E
9
B
~S
o z
AT AFTER
pH
OF
STANDRRD
pH
INDICATED
REROJUSTNENT
FOLIRR
TO p H 7
x 9
EXTRACT
FIG. 2. Stability of nematode attractiveness of aqueous extract of Solanum elaeagnifol(B) Partially irreversible loss of activity at low pH. Brackets indicate confidence intervals as in Figure 1,
ium foliage. (A) Partial loss of activity at 100~
488
ROBINSON
100
i
i
i
i
i
1
AND
SALDANA
i
7s
5~
_1
i.- cm z ~ r
I i-tu
z
::x
r :1:
25 T
9 EXTRRCT
-
A
x RESIDUE RE-EXTRBCTED WITH WRTER I
POLRRITT INDEX OF EXTRRCTION SOLUENT
EXTRRET
9
RESIDUE RE-EXTRRCTED WITH WRTER x
g01
4a.
20
~;
1
2 3 4S 6 7 8 POLRRITY INDEX OF EXTRRCTIDN SOLUENT
S
FIG. 3. (A) Effect of solvent polarity on extraction of attractants from dry Solanum elaeagnifolium foliage (2 % w/w). Each extract was filtered, evaporated to dryness, and reextracted with the original volume of water before bioassay. Residue from each filtration also was reextracted with the original volume of water, Brackets indicate confidence intervals as in Fig. 1. (B) Same information as in (A) expressed in terms of the percentage concentration of attractant in a dilution series of a standard aqueous extract (6% w/w) that was bioassayed concurrently. Note the exponential effect of attractant concentration on statistical uncertainty.
comprising the ring accumulated underneath the disks within 15 min. Measurements of agar pH various distances from the disk with a 2-ram micro-pH electrode indicated that pH elevation to about 4 was required for aggregation. This effect may result from repellents or attractants that are pH reversible in activity. Passage of 67 N SFE through anion ( C I - ) and cation (H + ) exchange columns lowered pH to ca. 2 and removed activity. Upon readjustment to pH 7, extract from the anion exchange column was strongly attractive and extract from the cation exchange column was unattractive (Table 1). Elution with 100 m M
Distilled water wash to condition column 67 % standard foliar extract (sample) Distilled water wash 100 m M KNO3 1 M KNO 3
1
9 4 4
3
4
46-60 48-62 27-41
69-81
38-48
Nematode response index (%)a
1 2 0
50
0
Apparent attractant concentration (%)b
47-60 55-68 29-42
52-65
43-57
1 5 0
3
1
Apparent attractant concentration (%)
Cation Exchange resin (H § AG 50W-X8) Nematode response index (%)
a Confidence limits (P = 0.05) of arcsine-transformed data, expressed as nematode response index as defined in text. OApparent concentration of attractant relative to standard foliar extract (6% w/w).
3 4 5
2
Eluant
Eluate
Eluant volume (ml)
Anion exchange resin (CI-, AG l-X8)
TABLE 1. NEMATODE ATTRACTANT IN ELUATES FROM POLY-PREP COLUMNS CONTAINING ION EXCHANGE RESINS AFTER LOADING WITH 3 - m l AQUEOUS EXTRACT PREPARED FROM Solanum elaeagnifolium FOLIAGE
4~ oo
490
ROBINSON AND SALDANA
KNO3 followed by adjustment to pH 7 yielded weak activity from the cation but not from the anion exchange resin. Therefore, attractant appeared completely unretained by anion exchange. Failure to recover full activity in the 100 mM KNO 3 cation exchange eluate may have resulted from incomplete elution, acid hydrolysis of attractants, or assay interference from NO~-. Both 1 M KNO 3 eluates were repellent. Toward HPLC Purification. High levels of activity were detected in the 6to 10- (I) and 31- to 35- (II) min fractions obtained with the C18 Semi-Prep column. Similar activity occurred in the corresponding fractions of the Sep-Pak cleanup (Figure 4). Bioassay results for subfractions of II from the analytical C18 cartridge showed activity in a baseline separated peak eluting at 9.0 min. Fraction I was evaporated to dryness and reextracted with original volumes of CH3CN-water mixtures. These were bioassayed, and the solubility of activity (Figure 5) was used to calculate appropriate concentrations of subfractions obtained for bioassay from the amine-bonded column. Most activity occurred in three distinct fractions (Figure 6), two of which contained consistently identifiable absorbance peaks at 230 and 254 nm. Absorbance maxima of active fractions occurred at 203 nm, where solvent interference prevented HPLC detection. The first strongly active fraction to elute induced a mean nematode response equivalent to 34% SFE. The material injected was 100/~1 of I in 90:10 CH3CN in water with an activity concentration equivalent to ca. 12 % SFE (Figure 5), and fractions were assayed at a final volume of 25 /~1. Therefore, the first active fraction to elute accounted for most of the activity injected. Known Compounds. Several compounds were weakly attractive, but even the strongest response, to cr was weaker than to 5 % SFE. Pyridine was strongly repellent (Table 2). Pyridine attracts the bacteriophagous nematode Caenorhabditis elegans (Dusenbery, 1976). Extraction of the foliar powder specifically for solanaceous glycoalkaloids, using methods established for potato, yielded products that were neither attractive nor repellent at neutral pH. Our results indicate that several substances within S. elaeagnifolium foliar extract attract infective juveniles of O. phyllobia in vitro. They appear to be similarly nonvolatile, dialyzable, polar, ionizable, water soluble, sensitive to very low pH, and stable at biological temperatures. We do not know if common functional groups are involved. Small, stable, water-soluble compounds from foliage would seem ideal for attracting nematodes within soil to plant stem bases. Nematodes, in general, move in water films, and optimum physical conditions for locomotion in soil occur at high water contents, near the inflection point of the soil moisture release curve (Wallace, 1968). We cannot be certain that our attractive HPLC fractions contain compounds that naturally occur in behaviorally active concentrations near living plants; we know nothing concerning their anatomical compartmentalization and occurrence in natural foliar leachates. Our approach has been to develop chromatographic methods for
NEMATODE
ATTRACTANTS
491
A I
II
A
Ti f
4O
B
I FRACTIONS OF AOUEOUSEXTRACT FROM C18 CLEAN-UP
~: 30
20
w o~ SAMPLE WATER NRTER 38:~0 .~ .3 .8S 1.S
t'tEOH .7
SOLUENT RATIOS OF METHANOL:WATER
tIEOH 50:SI] .5 2
URTER 2
RNO ELURTE UOLUMES
(ml)
FIG. 4. Preliminary HPLC separation of strong (I) and weak (II) sources of activity in aqueous extract of Solanum elaeagniflium foliage using a water-to-methanol gradient on C~8 Semi-Prep column, and assay results for eluates obtained through a C=8 Sep Pak cleanup procedure using solvent volumes that approximated the HPLC gradients. (A) UV absorbance for HPLC and locations of activity among eleven 5-min fractions. Solvent concentrations described in text. (B) Nematode response to fractions comparable to the HPLC fractions but obtained with the cleanup procedure. The tops of black and white bars for each fraction indicate the confidence limits (P = 0.05) of nematode response expressed as the equivalent percentage concentration of an extract dilution series assayed concurrently as described in Figure 3B.
detecting attractive substances before trying to find them in soil. Further research toward that goal is underway. The potato cyst nematode, Globodera rostochiensis, is a serious agronomic pest in potato production. It is ecologically similar to O. phyllobia as a nematode closely associated with Solanum spp. hosts. Since 1922, considerable effort has been put toward isolation and identification of a factor in root leachates from Solanum spp. that stimulates G. rostochiensis egg hatching. The active factor, which is still unidentified, chromatographs as several compounds that
492
ROBINSONAND SALDANA so
W E F-
i
i
i
r
~
i
i
~
~TIO~
i
i
t
OF F R ~ T I O N
I
40
rr h O Z O
30
er
E ao
m u z o u
i--- lO z w c~ fro.. o_ o
I
I
I
I0
20
I
30
I
I
I
I
I
I
40
SO
60
70
80
SO
PERCENTRGE
CH3EN
IN
/ 0
I0
WRTER
FIG. 5. The apparent concentration of attractant, relative to standard aqueous extract of
dry Solanum elaeagnifolium foliage (6 % w/w), in CH3CN-water extracts of dry fraction I. Fraction I was obtained by a Cls Sep Pak cleanup procedure, then evaporated to dryness and reextracted with an original volume of the solvent mixture indicated. Note that the concentration of attractant in 90% CH3CN is only 30% of the concentration in water. Brackets indicate confidence intervals as in Figure 3B.
UU R B S O R B R N E E RT 254 NM
so
4a
'7
3o
20 U
NEMATODE RESPONSE
E lo
w
S I0 IS 20 2S E L U T I O N T I M E OF S U B F R R E T I O N S OF F R R E T I O N
30 I (MINUTES)
3S
FIG. 6. Nematode attractant activity in 35 1-min HPLC subfractions of fraction I obtained with amine-bonded column and a CH3CN-water mobile-phase gradient. Solvent concentrations described in text. Bars indicate confidence intervals as in Figure 4B. The strongly attractive fraction eluting at 7-8 min accounted for 75 % of the total activity in the injected material.
493
NEMATODE ATTRACTANTS
TABLE 2. KNOWN COMPOUNDS TESTED FOR EFFECTS ON BEHAVIOR OF
Orrina
phyllobia
Compound cAMP L-Tryptophan D-Tryptophan Pyridine c~-Chaconine c~-Solanine Gitoxin Saponin Digitonin NaC1 NaOH KC1 KOH SFE 100 % 33 % 10% 3% 1% Distilled water
pH
Nematode response index confidence limits (%)a
Apparent concentration of attractant (% of SFE) b
30 10 25 25 25 25
7.2 5.8 6.0 3.2 7.7 7.4 6.2 6.1 5.8 6.3 11.5 5.9 11.9
49-63 51-66 56-70 16-27 d 59-73 47-62 44-59 57-71 40-55 53-68 53-68 58-72 51-65
1 2 3 0 5 1 1 3 0 2 2 4 1
98e 34e 11e 5e 1e
6.3 6.3 6.3 6.3 6.3
85-91 80-87 66-75 57-66 53-62 46-55
87 44 8 2 1 0
Concentration (mM) 20 20 20 150 c c c
aConfidence limits (P = 0.05) calculated from arcsine-transformed data for five replicates, then reconverted to nematode response index units as defined in text. bObtained by comparing the average nematode response index for five replicates to a response curve obtained concurrently for five dilutions of SFE spanning two orders of magnitude. SFE = standard foliar extract (6 % w/w, aqueous). CSaturated solutions < 4 mM by freezing point depression. dSignificantly repellant at P = 0.01. eOsmotic pressure (mM) by freezing point depression.
are h i g h l y p o l a r a n d w a t e r s o l u b l e in c h a r a c t e r ( A t k i n s o n et al., 1987). B e f o r e i n i t i a t i n g c h a r a c t e r i z a t i o n o f t h e O. phyllobia a t t r a c t a n t s , w e s p e c u l a t e d t h a t t h e y m i g h t b e s o l a n a c e o u s g l y c o a l k a l o i d s o r m i g h t b e r e l a t e d to t h e p o t a t o cyst n e m a t o d e h a t c h i n g factor. O u r o b s e r v a t i o n s s u p p o r t n e i t h e r p r e d i c t i o n . Satur a t e d s o l u t i o n s o f t h e c o m m o n Solanum g l y c o a l k a l o i d s c~-chaconine a n d ees o l a n i n e d i d n o t a t t r a c t O. phyllobia a p p r e c i a b l y ; e x t r a c t i o n specifically f o r glyc o a l k a l o i d s d i d n o t y i e l d a t t r a c t i v e p r o d u c t s ; a n d a t t r a c t i o n to S F E o c c u r r e d at a l k a l i n e p H s ( > 10), at w h i c h m a n y Solanum g l y c o a l k a l o i d s p r e c i p i t a t e f r o m
494
ROBINSON AND SALDANA
water. Recent work on potato cyst nematode hatching factor has shown it to be anionically but not cationically exchangeable (Atkinson et al., 1987); the converse was observed for O. phyllobia attractant. Acknowledgments--We thank Dr. Roger Albach (Agricultural Research Service, USDA) for valuable discussion and advice during the course of this study.
REFERENCES ALLEN, E.H., and Kuc, J. 1968. a-Solanine and c~-chaconineas fungitoxic compounds in extracts of Irish potatoes tubers. Phytopathology 58:776-781. ATKINSON, H.J., FOWLER, M., and ISAAC, R.E. 1987. Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate. Ann. Appl. Biol. 110:115-125. BALAN, J., KRIZKOVA,L., NEMEC, P., and KOLOZSVARY,A. 1976. A qualitative method for detection of nematode attracting substances and proof of production of three different attractants by the fungus Monacrosporium rutgeriensis. Nematologica 22:306-311. BUSHWAY, R.J., BUREAU,J.L., and ST1CKNEY, M. 1985. A new efficient method for extracting glycoalkaloids from dehydrated potatoes. J. Agric. Food Chem. 33:45-46. CARMAN, A.S. Jr., KUAN, S.S., WARE, G.M., FRANCIS, O.J. JR., and KIRSCHENHEUTER, G.P. 1986. Rapid high-performance liquid chromatographic determination of the potato glycoalkaloids a-solanine and c~-chaconine. J. Agric. Food Chem. 34:279-282. CROLL, N.A. 1970. The Behaviour of Nematodes, Their Activity, Senses, and Responses. St. Martin's Press, New York. 177 pp. DUSENBERV, D.B. 1976. Attraction of the nematode Caenorhabditis elegans to pyridine. Comp. Biochem. Physiol. 53C:1-2. DUSEtqBERV,D.B. 1983. Chemotactic behavior of nematodes. J. Nematol. 15:168-173. FUTAI, K. 1980. Host preference of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) and B. mucronatus shown by their aggregation to pine saps. Appl. Entomol. Zool. 15:193197. GREEN, C.D. 1971. Mating and host finding behavior of plant nematodes, pp. 247-266, in B.M. Zuckerman, W.F. Mai, and R.A. Rohde (eds.). Plant Parasitic Nematodes. Academic Press, New York. KLINGER, K. 1965. On the orientation of plant nematodes and of some other soil animals. Nematologica 11:4-18. MmLER, P.M., and McENTYRE, J.L. 1976. Activated charcoal inhibits the entry of Pratylenchus penetrans and Heterodera tabacum into tomato and tobacco roots. J. Nematol. 8:297-298. PEACOCK, F.C. 1961. A note on the attractiveness of roots to plant parasitic nematodes. Nematologica 6:85-86. PROT, J.C. 1980. Migration of plant-parasitic nematodes towards plant roots. Rev. Nematol. 3:305318. PYE, A.E., and BURMAN,M. 1981. Neoaplectana carpocapsae: Nematode accumulations on chemical and bacterial gradients. Exp. Parasitol. 51:13-20. ROmNSON, A.F., ORR, C.C., and ABERNATHV,J.R. 1978. Distribution of Nothanguina phyllobia and its potential as a biological control agent for silver-leaf nightshade. J. Nematol. 10:362366. ROBINSON, A.F., ORR, C.C., and ABERNATHY,J.R. 1979. Behavioral response of Nothanguina phyllobia to selected plant species. J. Nematol. 11:73-77.
NEMATODE ATTRACTANTS
495
STEINER, G. 1925. The problem of host selection and host specialization or certain plant-infesting nemas and its application in the study of nemic pests. Phytopathology 15:499-534. TOMINAGA, Y., NAGASE,A., KUWAHARA,Y., and SUGAWARA,R. 1983. Behavioral responses of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) to bitter and pungent substances. Appl. EntomoL Zool. 18:106-110. TOMINAGA, Y., YAMAMOTO,M., KUWAHARA,Y., and SUGAWARA,R. 1984. Behavioral responses of the pine wood nematode to terpenes. Agric. Biol. Chem. 48:519-520. WALLACE,H.R. 1968. The dynamics of nematode movement. Annu. Rev. Phytopathol. 6:91-114. WALLACE,H.R. 1973. Nematode Ecology and Plant Disease. Edward Arnold Ltd., London. 228 PP.
WARD, J.B., NORDSTROM,R.M., and BONE, L.W. 1984. Chemotaxis of male Nippostrongylus brasiliensis (Nematoda) to some biological compounds. Proc. Helminthol. Soc. Wash. 51:7377. WARD, S. 1973. Chemotaxis by the nematode Caenorhabditis elegans: Identification of attractants and analysis of the response by use of mutants. Proc. Natl. Acad. Sci. U.S.A. 70:817-821. ZUCKERMAN,B.M., and JANSSON, H.B. 1984. Nematode chemotaxis and possible mechanisms of host/prey recognition. Annu. Rev. Phytopathol. 22:95-113.
CHARACTERIZATION A N D PARTIAL PURIFICATION OF ATTRACTANTS FOR NEMATODE Orrina phyllobia FROM FOLIAGE OF Solanum elaeagnifolium
A.F. ROBINSON
A N D G. S A L D A N A
Agricultural Research Service, USDA Subtropical Agricultural Research Laboratory Weslaco, Texas 78596 (Received November 9, 1987; accepted January 8, 1988) Abstract--An unknown attractant for the nematode Orrina phyllobia was extracted with water from foliage of Solanum elaeagnifolium. Stability, solubility, ionic character, and chromatographic purification were investigated using a bioassay based on nematode aggregation in agar. Activity was nonvolatile, dialyzable, heat stable below 60~ and partially lost within 30 min at 100~ Activity was unchanged from pH 5 to 12, but was entirely lost at pH 2. Loss of activity at low pH did not appear to result from direct effects of pH on nematode behavior and was partially recovered by readjustment to pH 7. The attractive factor was most soluble in water and appeared to be cationically but not anionically exchangeable. Activity appeared to chromatograph as several compounds by high-performance liquid chromatography employing reverse phase C~8 and amine-bonded columns. Various known compounds that are common to Solanum spp. or that attract other nematodes were unattractive. Extraction of S. elaeagnifolium foliage specifically for solanaceous glycoalkaloids using methods developed for S. tuberosum did not yield an attractive product. Key Words--Orrina phyllobia, Solanum elaeagnifolium, foliar nematode, host finding, nematode chemotaxis, silverleaf nightshade.
INTRODUCTION
M o r e than 4 0 0 0 n e m a t o d e species parasitize plants and considerable research has b e e n d o n e t o w a r d learning h o w s o i l - b o r n e i n f e c t i v e stages find roots and stems ( r e v i e w e d by Steiner, 1925; Croll, 1970; G r e e n , 1971; Prot, 1980; Z u c k e r m a n and J a n s s o n , 1984). N e m a t o d e orientation to t e m p e r a t u r e and soil m o i s 481
482
ROBINSON AND SALDANA
ture gradients may assist host finding, but most evidence indicates chemotaxis is primarily responsible. Very little is known concerning specifically phytogenic attractants. Many nematodes are attracted to root leachates; in at least two cases, root leachate attractiveness was removed with activated charcoal, suggesting an organic nature (Miller and Mclntyre, 1976; Peacock, 1961). Various common biogenic chemicals, largely amino acids, attract or repel some bacteriophagous, phytoparasitic, and zooparasitic nematodes on agar or agarose gels (Dusenbery, 1983; Pye and Burman, 1981; J.B. Ward et al., 1984; S. Ward, 1973). These include L-tryptophan, glutamic acid, aspartic acid, cysteine, L-tyrosine and other amines, cAMP, cGMP, gibberellic acid, glutathione, ascorbic acid, and pyridine. These responses were discovered by screening hundreds of known compounds and none has been established to mediate orientation within soil (Dusenbery, 1983). Failure to identify plant-specific attractants has encouraged the view (Klingler, 1965; Prot, 1980; Wallace, 1973) that phytoparasitic nematodes in soil may rely primarily on nonspecific chemical stimuli, such as CO2 and inorganic ions for orientation. Several species orient on gradients of CO2, Na +, CI-, or O H - . Progress toward understanding nematode chemotaxis as it relates to specific plant hosts has been made with the pine-wood nematode, Bursaphelenchus xylophilus (Futai, 1980). Various terpenes and other compounds common to the coniferous plants are attractive or repellant in vitro (Tominaga et al., 1983, 1984). The life cycle of this nematode differs from most phytoparasitic species in that it is normally transmitted to the trunk and branches of the host tree by a pine sawyer beetle (Monochamus alternatus), rather than by moving through soil. Thereafter, nematodes move and feed within resin canals where behavioral responses to terpenes may play an important role. Chemotactic preferences for tree saps appear related to host suitability among tree genera but not among pine species (Futai, 1980). A second nematode whose behavior appears attuned to specific plants is Orrina phyllobia. It is known to parasitize only foliar parts of certain plants within the genus Solanum and is specifically attracted to leachates from stems of four Solanum spp. These are S. tuberosum (potato), S. melongena (eggplant), S. carolinense (Carolina horse nettle), and S. elaeagnifolium (silverleaf nightshade). Leachates from rhizomes of Solanum spp. and from stems of 30 plant species in other genera spanning 12 plant families were not attractive (Robinson et al., 1979). Attraction to stems is ecologically valuable to O. phyllobia because its life cycle entails aggregation of infective juveniles, which are about 700 tzm long, within soil around stem bases and emerging shoot tips. When stem surfaces are moist, the juveniles ascend as far as 50 cm and invade foliar meristems to initiate new infections (Robinson et al., 1978). We have begun to characterize and chromatographically purify attractant from foliage of the common host,
483
NEMATODE ATTRACTANTS
S. elaeagnifolium, with a view toward understanding this rather host-specific behavior by a phytoparasitic nematode.
METHODS AND MATERIALS
Bioassay Procedure. Stems and leaves of S. elaeagnifolium were collected from field-grown plants, freeze dried intact, and ground with a Wiley mill. The powder obtained was stored in a desiccator over anhydrous CaSO 4 and used as a standard source of attractant. Preparations from plants collected on other occasions were similarly attractive. The assay we adopted was a modification of methods described by Balan et al. (1976) and Tominaga et al. (1983) for studying chemotaxis in other nematodes. Infective juveniles of O. phyllobia were removed daily from dried leaf galls of S. elaeagnifolium as previously described (Robinson et al., 1979) and suspended in a continuously aerated dilute salt solution (4.5 mM NaC1, 0.4 mM KC1, 0.05 mM CaC12, 0.05 mM MgC12) to reduce ion regulation stress. Bacto-Agar (1.5%, Difco) was dissolved in water (w/w) supplemented with dilute salts equivalent to those in the nematode suspension. Equal parts of nematode suspension (22 ~ and agar (42 ~C) were then mixed and poured into a 35-ram-diameter Petri dish to obtain a 2-ram-deep film of 0.75 % agar containing about 3000 juveniles. The mixing procedure induced a brief reduction in motility from which nematodes completely recovered within several minutes. A 3-mm-diameter disk of filter paper (Whatman No. 1) saturated with the test solution and air-dried was placed halfway between the center and the edge of the dish. Trials with aqueous solutions of the biological stain safranin-O indicated that the pattern of diffusion of material from the dried disk was hemispherical downward. Two untreated disks were then placed equidistant from the center, 1 cm from each side of the treated disk. After 90 min, a 5-ram cork borer and small spatula were used to transfer agar cylinders containing the disks to counting dishes where the nematodes within each cylinder were dispersed into water and microscopically counted. The response index (RI) was defined as RI = T/(T + U) where T = nematodes under the treated disk and U = average number of nematodes under untreated disks in the same dish. RI > 50 % indicated aggregation and RI < 50 %, repulsion. RI values were binomially distributed and examined statistically after arcsine transformation. In each experiment except where noted, five dilutions spanning two orders of magnitude were prepared for bioassay from filtered aqueous extract of the dry plant powder (6 % w/w). The 6 % concentration will be referred to as standard foliar extract (SFE). Nematode chemotaxis experiments are frequently conducted without light to mimic soil conditions. In preliminary experiments, we found that bioassay
484
ROBINSON AND SALDANA
results obtained under laboratory light were identical to results obtained in the dark. Small temperature gradients (ca. 0.1 ~ that resulted from differential evaporation of water within dishes strongly influenced nematode distributions. However, dishes were covered and oriented identically to minimize the effect. We recognized that test solutions might generate behaviorally important extremes of pH, ionic strength, and osmotic potential. To evaluate their contributions, we bioassayed aqueous solutions of acetic acid, HC1, and NaOH at 0.01, 0.1, 1, 10, and 100 raM. We also bioassayed solutions with osmotic pressures from 100 to 500 mM by supplementing water and foliar extract with sucrose or inorganic ions in ratios described. Standard foliar extract had 100120 mM osmotic pressure by freezing point depression and pH 6.3-6.5. Bioassays were performed in five replicates. Experiments were repeated at least once. Reproducibility was sometimes confirmed by assigning numerical ratings to aggregations instead of counting nematodes. Preliminary Characterization of Attractants. Standard foliar extract was bioassayed after each of the following procedures: dialysis against distilled water across 3500 MWCO dialysis tubing; filtration through activated charcoal; heating to 50, 60, 70, 90, and 100~ for various intervals up to 75 min; pH adjustment with HC1/NaOH; and readjustment to pH 7. Extract had appreciable buffering capacity near pH 6.5, and neutralization beyond 3 pH units required excessive dilution of extract or increases in Na § and C1- concentrations > 200 raM. These are physiologically significant levels; therefore, control solutions were prepared with similar osmotic pressures by adding NaC1 to extract without pH adjustment. To examine attractant solubility, foliar powder was extracted (2 % w/w) with solvent mixtures in a polarity series including water, methanol, 95% ethanol, 1-propanol, 1-butanol, diethyl ether, and hexane. Each extract was then filtered, evaporated to dryness, and reextracted with water at original volume. Residue obtained from the first filtration was separately reextracted with water and all solutions assayed. Ionic character was examined with prefilled Poly-Prep columns (Bio-Rad) containing 2 ml of AG 50W-X8 and AG l-X8 ion exchange resins in the H + and C1- forms. Aqueous extract was prepared at 67% the concentration of SFE. After washing with distilled water, resin was drained under vacuum, and 3 ml of extract was passed through each column. Columns were then washed with 9 rnl distilled water and eluted with 4 ml 100 rnM KNO3, followed by another 4 ml 1 M KNO3. Eluates and washes were bioassayed before and after adjustment to pH 7 with NaOH. Toward HPLC Purification. Preliminary separations were made with a Waters HPLC system equipped with a reverse phase Cls Semi-Prep column (7.8 m m x 30 cm) (Waters) and a variable wavelength detector set at 254 nm. Solvent flow was 1.5 ml/min as follows: 15 rain isocratic water, 10 min linear gradient to 100% methanol, 10 rain hold, 20 rain linear gradient to 100% water. Eleven 5-rain fractions were collected separately from three 50-/zl injections of
NEMATODE ATTRACTANTS
485
67% SFE, composited, evaporated to dryness (60~ redissolved with 2 ml water, then reevaporated to dryness in small dishes under vacuum (50~ and finally redissolved in 150 #1 water for bioassay. Solvent controls were also tested. A cleanup procedure using disposable C~8 preparatory columns (Sep Pak, Waters) was then developed based on the column volume equivalents of eluants needed for a stepwise approximation to the HPLC solvent gradient. Active fractions were prepared in quantity with the cleanup procedure. Further separations were done with an analytical C18 Radial Pak cartridge (8 mm x 10 cm) or an amine-bonded column (3.9 mm x 30 cm) (Waters). Mobile phase for the C18 cartridge was a 25-min water-methanol linear gradient ( 1.0 ml/min) from 20 % to 80 % methanol. Mobile phase for the amine-bonded column was a 20-min CH3CN-water linear gradient (2.0 ml/min) from 90% to 65% CH3CN, a 5-min hold, and a linear gradient back to inititial conditions. Thirtyfive 1-min fractions were collected separately from two injections, composited, evaporated, reextracted with 25 ~1 water, and bioassayed. Examination of Known Substances for Activity. Commercial preparations (Sigma) of various compounds were bioassayed because they are associated with Solanum spp. or because they have chemotactic activity to other nematodes. These included the common solanaceous glycoalkaloids c~-chaconine and o~-solanine at pH 7.5. With the view that attractants might occur among other Solanum-specific glycoalkaloids, the procedures described by Allen and Kuc (1968), by Bushway et al. (1985), and by Carman et al. (1986) for extracting glycoalkaloids from potato were applied to the S. elaeagnifolium foliar powder and confirmed by HPLC of product against commercial preparations of o~-chaconine and a-solanine.
RESULTS AND DISCUSSION
Bioassay Procedure. The RI reached equilibrium after ca. 60 min (Figure 1A). The RI always approximated a log decay function of concentration that asymptotically approached 85-95 %. The least-squares line relating R1 and log10 concentration was employed to express bioassay results in terms of the apparent concentration of attractant as well as in RI units (Figure 1B). Results of one experiment (Figure 3 below) are presented in both ways to convey the exponential relationship between attractant concentration and statistical uncertainty. Independent effects of pH on the bioassay were not detected with acetic acid and HC1 solutions of pH > 2. NaOH > 10 mM caused weak aggregation and at pH 12 was equivalent to the effect of 3% SFE. Adding sucrose or mixed salts to water and foliar extract to generate osmotic pressures as high as 500 mM did not change bioassay results. Osmotic pressures of test solutions in subsequent experiments were < 300 mM. Some species of nematodes respond
486
ROBINSON AND SALDANA i
A 7s
z REPULSION
25
RESPONSE TO STANDARD FOLIAR EXTRACT
2'o
,'o
2~
8'~
DURATION OF ASSAY (MINUTES1 101~
B
i
i
J
t
~ 6a
CONCENTRATION (kOGlo STANDARD FOLIAR EXTRACT)
FIo. 1. Effects of bioassay duration and foliar attractant concentration on the nematode response index. (A) Increase in nematode response during 90-min exposure to aqueous extract of Solanum elaeagnifolium foliage. (B) Representative concentration-response curve for aqueous foliar extract. Concentrations are expressed as percentage of a standard concentration of dry foliage in water (6 % w/w). Brackets indicate confidence intervals for five replicates obtained after arcsine transformation. The straight line and the sigmoid log decay function [dy/dx = (Y - y)K, where y = response index, x = concentration, Y = the maximum value of y, and K = a constant] were fitted by least squares.
behaviorally to pH, ion, and osmotic pressure gradients much smaller than we imposed (Dusenbery, 1983; Prot, 1980). I f O. phyllobia also responds to them, their effects are very small under our assay conditions compared with the effects o f attractant within S. elaeagnifolium foliage. Preliminary Characterization of Attractants. Attractant from S. elaeagnifolium was essentially nonvolatile, freely dialyzable, and heat stable for 90 min under the conditions we u s e d for evaporating test solutions ( < 6 0 ~ How-
NEMATODE
487
ATTRACTANTS
ever, some activity was lost within 30 min at 100~ (Figure 2A). Further losses were not detected up to 90 rain, suggesting that heating generated a repulsive product or that two or more attractants were present and differed in temperature sensitivity. Activity was highly polar or ionic in character, not removed with activated charcoal, and most soluble in water (Figure 3). The attractiveness of standard fotiar extract was completely lost by adjusting pH to 2 with HC1 but was partially recovered after readjustment to 7 (Figure 2B). Addition of NaC1 to standard foliar extract at concentrations equal to those generated by pH adjustment did not reduce activity, suggesting that direct effects of Na + and C1- on nematode behavior were not important factors. Disks with SFE at low pH frequently induced nematodes to aggregate in a halolike ring surrounding the disk 2-4 mm from its edge. When a second disk, which had been saturated with 0.1 N NaOH and dried, was placed on top of the first disk, the nematodes J
,
,
A
ATTRACTION ................................................ REPULSION
60 i00
TINE
THAT
J
EXTRRCT
WAS
i
HEATED
(NINUTES)
i
J
C
•
E
9
B
~S
o z
AT AFTER
pH
OF
STANDRRD
pH
INDICATED
REROJUSTNENT
FOLIRR
TO p H 7
x 9
EXTRACT
FIG. 2. Stability of nematode attractiveness of aqueous extract of Solanum elaeagnifol(B) Partially irreversible loss of activity at low pH. Brackets indicate confidence intervals as in Figure 1,
ium foliage. (A) Partial loss of activity at 100~
488
ROBINSON
100
i
i
i
i
i
1
AND
SALDANA
i
7s
5~
_1
i.- cm z ~ r
I i-tu
z
::x
r :1:
25 T
9 EXTRRCT
-
A
x RESIDUE RE-EXTRBCTED WITH WRTER I
POLRRITT INDEX OF EXTRRCTION SOLUENT
EXTRRET
9
RESIDUE RE-EXTRRCTED WITH WRTER x
g01
4a.
20
~;
1
2 3 4S 6 7 8 POLRRITY INDEX OF EXTRRCTIDN SOLUENT
S
FIG. 3. (A) Effect of solvent polarity on extraction of attractants from dry Solanum elaeagnifolium foliage (2 % w/w). Each extract was filtered, evaporated to dryness, and reextracted with the original volume of water before bioassay. Residue from each filtration also was reextracted with the original volume of water, Brackets indicate confidence intervals as in Fig. 1. (B) Same information as in (A) expressed in terms of the percentage concentration of attractant in a dilution series of a standard aqueous extract (6% w/w) that was bioassayed concurrently. Note the exponential effect of attractant concentration on statistical uncertainty.
comprising the ring accumulated underneath the disks within 15 min. Measurements of agar pH various distances from the disk with a 2-ram micro-pH electrode indicated that pH elevation to about 4 was required for aggregation. This effect may result from repellents or attractants that are pH reversible in activity. Passage of 67 N SFE through anion ( C I - ) and cation (H + ) exchange columns lowered pH to ca. 2 and removed activity. Upon readjustment to pH 7, extract from the anion exchange column was strongly attractive and extract from the cation exchange column was unattractive (Table 1). Elution with 100 m M
Distilled water wash to condition column 67 % standard foliar extract (sample) Distilled water wash 100 m M KNO3 1 M KNO 3
1
9 4 4
3
4
46-60 48-62 27-41
69-81
38-48
Nematode response index (%)a
1 2 0
50
0
Apparent attractant concentration (%)b
47-60 55-68 29-42
52-65
43-57
1 5 0
3
1
Apparent attractant concentration (%)
Cation Exchange resin (H § AG 50W-X8) Nematode response index (%)
a Confidence limits (P = 0.05) of arcsine-transformed data, expressed as nematode response index as defined in text. OApparent concentration of attractant relative to standard foliar extract (6% w/w).
3 4 5
2
Eluant
Eluate
Eluant volume (ml)
Anion exchange resin (CI-, AG l-X8)
TABLE 1. NEMATODE ATTRACTANT IN ELUATES FROM POLY-PREP COLUMNS CONTAINING ION EXCHANGE RESINS AFTER LOADING WITH 3 - m l AQUEOUS EXTRACT PREPARED FROM Solanum elaeagnifolium FOLIAGE
4~ oo
490
ROBINSON AND SALDANA
KNO3 followed by adjustment to pH 7 yielded weak activity from the cation but not from the anion exchange resin. Therefore, attractant appeared completely unretained by anion exchange. Failure to recover full activity in the 100 mM KNO 3 cation exchange eluate may have resulted from incomplete elution, acid hydrolysis of attractants, or assay interference from NO~-. Both 1 M KNO 3 eluates were repellent. Toward HPLC Purification. High levels of activity were detected in the 6to 10- (I) and 31- to 35- (II) min fractions obtained with the C18 Semi-Prep column. Similar activity occurred in the corresponding fractions of the Sep-Pak cleanup (Figure 4). Bioassay results for subfractions of II from the analytical C18 cartridge showed activity in a baseline separated peak eluting at 9.0 min. Fraction I was evaporated to dryness and reextracted with original volumes of CH3CN-water mixtures. These were bioassayed, and the solubility of activity (Figure 5) was used to calculate appropriate concentrations of subfractions obtained for bioassay from the amine-bonded column. Most activity occurred in three distinct fractions (Figure 6), two of which contained consistently identifiable absorbance peaks at 230 and 254 nm. Absorbance maxima of active fractions occurred at 203 nm, where solvent interference prevented HPLC detection. The first strongly active fraction to elute induced a mean nematode response equivalent to 34% SFE. The material injected was 100/~1 of I in 90:10 CH3CN in water with an activity concentration equivalent to ca. 12 % SFE (Figure 5), and fractions were assayed at a final volume of 25 /~1. Therefore, the first active fraction to elute accounted for most of the activity injected. Known Compounds. Several compounds were weakly attractive, but even the strongest response, to cr was weaker than to 5 % SFE. Pyridine was strongly repellent (Table 2). Pyridine attracts the bacteriophagous nematode Caenorhabditis elegans (Dusenbery, 1976). Extraction of the foliar powder specifically for solanaceous glycoalkaloids, using methods established for potato, yielded products that were neither attractive nor repellent at neutral pH. Our results indicate that several substances within S. elaeagnifolium foliar extract attract infective juveniles of O. phyllobia in vitro. They appear to be similarly nonvolatile, dialyzable, polar, ionizable, water soluble, sensitive to very low pH, and stable at biological temperatures. We do not know if common functional groups are involved. Small, stable, water-soluble compounds from foliage would seem ideal for attracting nematodes within soil to plant stem bases. Nematodes, in general, move in water films, and optimum physical conditions for locomotion in soil occur at high water contents, near the inflection point of the soil moisture release curve (Wallace, 1968). We cannot be certain that our attractive HPLC fractions contain compounds that naturally occur in behaviorally active concentrations near living plants; we know nothing concerning their anatomical compartmentalization and occurrence in natural foliar leachates. Our approach has been to develop chromatographic methods for
NEMATODE
ATTRACTANTS
491
A I
II
A
Ti f
4O
B
I FRACTIONS OF AOUEOUSEXTRACT FROM C18 CLEAN-UP
~: 30
20
w o~ SAMPLE WATER NRTER 38:~0 .~ .3 .8S 1.S
t'tEOH .7
SOLUENT RATIOS OF METHANOL:WATER
tIEOH 50:SI] .5 2
URTER 2
RNO ELURTE UOLUMES
(ml)
FIG. 4. Preliminary HPLC separation of strong (I) and weak (II) sources of activity in aqueous extract of Solanum elaeagniflium foliage using a water-to-methanol gradient on C~8 Semi-Prep column, and assay results for eluates obtained through a C=8 Sep Pak cleanup procedure using solvent volumes that approximated the HPLC gradients. (A) UV absorbance for HPLC and locations of activity among eleven 5-min fractions. Solvent concentrations described in text. (B) Nematode response to fractions comparable to the HPLC fractions but obtained with the cleanup procedure. The tops of black and white bars for each fraction indicate the confidence limits (P = 0.05) of nematode response expressed as the equivalent percentage concentration of an extract dilution series assayed concurrently as described in Figure 3B.
detecting attractive substances before trying to find them in soil. Further research toward that goal is underway. The potato cyst nematode, Globodera rostochiensis, is a serious agronomic pest in potato production. It is ecologically similar to O. phyllobia as a nematode closely associated with Solanum spp. hosts. Since 1922, considerable effort has been put toward isolation and identification of a factor in root leachates from Solanum spp. that stimulates G. rostochiensis egg hatching. The active factor, which is still unidentified, chromatographs as several compounds that
492
ROBINSONAND SALDANA so
W E F-
i
i
i
r
~
i
i
~
~TIO~
i
i
t
OF F R ~ T I O N
I
40
rr h O Z O
30
er
E ao
m u z o u
i--- lO z w c~ fro.. o_ o
I
I
I
I0
20
I
30
I
I
I
I
I
I
40
SO
60
70
80
SO
PERCENTRGE
CH3EN
IN
/ 0
I0
WRTER
FIG. 5. The apparent concentration of attractant, relative to standard aqueous extract of
dry Solanum elaeagnifolium foliage (6 % w/w), in CH3CN-water extracts of dry fraction I. Fraction I was obtained by a Cls Sep Pak cleanup procedure, then evaporated to dryness and reextracted with an original volume of the solvent mixture indicated. Note that the concentration of attractant in 90% CH3CN is only 30% of the concentration in water. Brackets indicate confidence intervals as in Figure 3B.
UU R B S O R B R N E E RT 254 NM
so
4a
'7
3o
20 U
NEMATODE RESPONSE
E lo
w
S I0 IS 20 2S E L U T I O N T I M E OF S U B F R R E T I O N S OF F R R E T I O N
30 I (MINUTES)
3S
FIG. 6. Nematode attractant activity in 35 1-min HPLC subfractions of fraction I obtained with amine-bonded column and a CH3CN-water mobile-phase gradient. Solvent concentrations described in text. Bars indicate confidence intervals as in Figure 4B. The strongly attractive fraction eluting at 7-8 min accounted for 75 % of the total activity in the injected material.
493
NEMATODE ATTRACTANTS
TABLE 2. KNOWN COMPOUNDS TESTED FOR EFFECTS ON BEHAVIOR OF
Orrina
phyllobia
Compound cAMP L-Tryptophan D-Tryptophan Pyridine c~-Chaconine c~-Solanine Gitoxin Saponin Digitonin NaC1 NaOH KC1 KOH SFE 100 % 33 % 10% 3% 1% Distilled water
pH
Nematode response index confidence limits (%)a
Apparent concentration of attractant (% of SFE) b
30 10 25 25 25 25
7.2 5.8 6.0 3.2 7.7 7.4 6.2 6.1 5.8 6.3 11.5 5.9 11.9
49-63 51-66 56-70 16-27 d 59-73 47-62 44-59 57-71 40-55 53-68 53-68 58-72 51-65
1 2 3 0 5 1 1 3 0 2 2 4 1
98e 34e 11e 5e 1e
6.3 6.3 6.3 6.3 6.3
85-91 80-87 66-75 57-66 53-62 46-55
87 44 8 2 1 0
Concentration (mM) 20 20 20 150 c c c
aConfidence limits (P = 0.05) calculated from arcsine-transformed data for five replicates, then reconverted to nematode response index units as defined in text. bObtained by comparing the average nematode response index for five replicates to a response curve obtained concurrently for five dilutions of SFE spanning two orders of magnitude. SFE = standard foliar extract (6 % w/w, aqueous). CSaturated solutions < 4 mM by freezing point depression. dSignificantly repellant at P = 0.01. eOsmotic pressure (mM) by freezing point depression.
are h i g h l y p o l a r a n d w a t e r s o l u b l e in c h a r a c t e r ( A t k i n s o n et al., 1987). B e f o r e i n i t i a t i n g c h a r a c t e r i z a t i o n o f t h e O. phyllobia a t t r a c t a n t s , w e s p e c u l a t e d t h a t t h e y m i g h t b e s o l a n a c e o u s g l y c o a l k a l o i d s o r m i g h t b e r e l a t e d to t h e p o t a t o cyst n e m a t o d e h a t c h i n g factor. O u r o b s e r v a t i o n s s u p p o r t n e i t h e r p r e d i c t i o n . Satur a t e d s o l u t i o n s o f t h e c o m m o n Solanum g l y c o a l k a l o i d s c~-chaconine a n d ees o l a n i n e d i d n o t a t t r a c t O. phyllobia a p p r e c i a b l y ; e x t r a c t i o n specifically f o r glyc o a l k a l o i d s d i d n o t y i e l d a t t r a c t i v e p r o d u c t s ; a n d a t t r a c t i o n to S F E o c c u r r e d at a l k a l i n e p H s ( > 10), at w h i c h m a n y Solanum g l y c o a l k a l o i d s p r e c i p i t a t e f r o m
494
ROBINSON AND SALDANA
water. Recent work on potato cyst nematode hatching factor has shown it to be anionically but not cationically exchangeable (Atkinson et al., 1987); the converse was observed for O. phyllobia attractant. Acknowledgments--We thank Dr. Roger Albach (Agricultural Research Service, USDA) for valuable discussion and advice during the course of this study.
REFERENCES ALLEN, E.H., and Kuc, J. 1968. a-Solanine and c~-chaconineas fungitoxic compounds in extracts of Irish potatoes tubers. Phytopathology 58:776-781. ATKINSON, H.J., FOWLER, M., and ISAAC, R.E. 1987. Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate. Ann. Appl. Biol. 110:115-125. BALAN, J., KRIZKOVA,L., NEMEC, P., and KOLOZSVARY,A. 1976. A qualitative method for detection of nematode attracting substances and proof of production of three different attractants by the fungus Monacrosporium rutgeriensis. Nematologica 22:306-311. BUSHWAY, R.J., BUREAU,J.L., and ST1CKNEY, M. 1985. A new efficient method for extracting glycoalkaloids from dehydrated potatoes. J. Agric. Food Chem. 33:45-46. CARMAN, A.S. Jr., KUAN, S.S., WARE, G.M., FRANCIS, O.J. JR., and KIRSCHENHEUTER, G.P. 1986. Rapid high-performance liquid chromatographic determination of the potato glycoalkaloids a-solanine and c~-chaconine. J. Agric. Food Chem. 34:279-282. CROLL, N.A. 1970. The Behaviour of Nematodes, Their Activity, Senses, and Responses. St. Martin's Press, New York. 177 pp. DUSENBERV, D.B. 1976. Attraction of the nematode Caenorhabditis elegans to pyridine. Comp. Biochem. Physiol. 53C:1-2. DUSEtqBERV,D.B. 1983. Chemotactic behavior of nematodes. J. Nematol. 15:168-173. FUTAI, K. 1980. Host preference of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) and B. mucronatus shown by their aggregation to pine saps. Appl. Entomol. Zool. 15:193197. GREEN, C.D. 1971. Mating and host finding behavior of plant nematodes, pp. 247-266, in B.M. Zuckerman, W.F. Mai, and R.A. Rohde (eds.). Plant Parasitic Nematodes. Academic Press, New York. KLINGER, K. 1965. On the orientation of plant nematodes and of some other soil animals. Nematologica 11:4-18. MmLER, P.M., and McENTYRE, J.L. 1976. Activated charcoal inhibits the entry of Pratylenchus penetrans and Heterodera tabacum into tomato and tobacco roots. J. Nematol. 8:297-298. PEACOCK, F.C. 1961. A note on the attractiveness of roots to plant parasitic nematodes. Nematologica 6:85-86. PROT, J.C. 1980. Migration of plant-parasitic nematodes towards plant roots. Rev. Nematol. 3:305318. PYE, A.E., and BURMAN,M. 1981. Neoaplectana carpocapsae: Nematode accumulations on chemical and bacterial gradients. Exp. Parasitol. 51:13-20. ROmNSON, A.F., ORR, C.C., and ABERNATHV,J.R. 1978. Distribution of Nothanguina phyllobia and its potential as a biological control agent for silver-leaf nightshade. J. Nematol. 10:362366. ROBINSON, A.F., ORR, C.C., and ABERNATHY,J.R. 1979. Behavioral response of Nothanguina phyllobia to selected plant species. J. Nematol. 11:73-77.
NEMATODE ATTRACTANTS
495
STEINER, G. 1925. The problem of host selection and host specialization or certain plant-infesting nemas and its application in the study of nemic pests. Phytopathology 15:499-534. TOMINAGA, Y., NAGASE,A., KUWAHARA,Y., and SUGAWARA,R. 1983. Behavioral responses of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) to bitter and pungent substances. Appl. EntomoL Zool. 18:106-110. TOMINAGA, Y., YAMAMOTO,M., KUWAHARA,Y., and SUGAWARA,R. 1984. Behavioral responses of the pine wood nematode to terpenes. Agric. Biol. Chem. 48:519-520. WALLACE,H.R. 1968. The dynamics of nematode movement. Annu. Rev. Phytopathol. 6:91-114. WALLACE,H.R. 1973. Nematode Ecology and Plant Disease. Edward Arnold Ltd., London. 228 PP.
WARD, J.B., NORDSTROM,R.M., and BONE, L.W. 1984. Chemotaxis of male Nippostrongylus brasiliensis (Nematoda) to some biological compounds. Proc. Helminthol. Soc. Wash. 51:7377. WARD, S. 1973. Chemotaxis by the nematode Caenorhabditis elegans: Identification of attractants and analysis of the response by use of mutants. Proc. Natl. Acad. Sci. U.S.A. 70:817-821. ZUCKERMAN,B.M., and JANSSON, H.B. 1984. Nematode chemotaxis and possible mechanisms of host/prey recognition. Annu. Rev. Phytopathol. 22:95-113.
