Journal of Chemical Ecology, Vol. 15, No. 6, 1989
ROOT-MEDIATED EFFECTS IN CARROT RESISTANCE TO THE CARROT FLY, P s i l a r o s a e
AMEL
M A K I and M . F . R Y A N Department of Zoology University College Dublin BeIfield, Dublin 4, Ireland
(Received March 21, 1988; accepted September 20, 1988) Abstract--Field experiments on two different soil types in Ireland assessed the extent and mechanisms of resistance to Psila rosae (F.), the carrot fly, with emphasis on the role of the carrot root. Ten carrot cultivars gave consistent results in terms of resistant and susceptible cultivars. Nonpreference oviposition was confirmed as a mechanism, and the use of egg traps, providing differential exposure of the main root, showed this was regulated by root factors, probably chemical constituents. Independent main root resistance to the larva was also confirmed, and this effect was established as consistent with a chemically mediated nonpreference. Antibiosis by the root was demonstrated. Such effects in three different modes indicate that main root properties are crucial in carrot resistance to Psila and suggest a pervasive influence of root chemicals on such resistance. Key Words--Carrot resistance, carrot fly, Psila rosae, Diptera, Psilidae, main root factors, nonpreference oviposition, root resistance to larvae, antibiosis, root chemicals.
INTRODUCTION T h e first report o f resistance in carrot (Daucus carota L.) against the carrot fly [Psila rosae (F.)] indicated n o n p r e f e r e n c e in e g g - l a y i n g as a contributing factor ( A h l b e r g , 1944). M o s t subsequent investigations e m p h a s i z e d screening for resistance, w h i c h led to the f o r m a t i o n in 1976 o f a w o r k i n g group o f the International O r g a n i s a t i o n for B i o l o g i c a l C o n t r o l ( I O B C ) with representatives f r o m nine E u r o p e a n countries. T h e i r data f r o m 12 sites demonstrated resistance as h a v i n g sufficient effect and c o n s i s t e n c y to suggest a role for resistant cultivars in the integrated control o f Psila (Ellis and H a r d m a n , 1981); h o w e v e r , the 1867 0098-0331/89/0600-1867506.00/0 9 1989 Plenum Publishing Corporation
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MAKI AND RYAN
mechanisms of resistance were not ascertained. These were examined in this laboratory, where we also addressed the role of chemical factors using a range of cultivars different from those of the IOBC (Ryan et al., 1978). Our field experiments confirmed nonpreference at the level of oviposition and detected a new effect in the form of root resistance to the larva that was independent of resistance to egg-laying (Guerin and Ryan, 1984); that is, two cultivars attracting equally few eggs could sustain widely different degrees of root damage. As such damage is the direct cause of crop losses, it follows that root resistance to the larva is a fundamental requirement in breeding resistance (Guerin and Ryan, 1984). It was not possible to decide if this operated through larval nonpreference or antibiosis, and this question is addressed in the present report. It was also clear that intact roots of resistant varieties released substantially less volatile material than susceptible ones as judged by comparison of steam distillates and of headspace vapors entrapped by Porapak Q (Guerin and Ryan, 1984). We now report on field experiments with the IOBC range of cultivars. The results confirm nonpreference at the level of oviposition, independent root resistance to the larva, and show, in addition, that the latter is consistent with a chemically mediated nonpreference effect. The results unambiguously demonstrate antibiosis as independently elicited by the root, which also involves chemicals. Finally, and perhaps surprisingly, they indicate that root factors, as distinct from foliage ones, can govern nonpreference oviposition. Collectively, such data establish the root as capable of regulating carrot resistance to Psila, probably through its chemical constituents.
METHODS AND MATERIALS
A total of five field experiments were conducted over three years to assess both the extent of resistance to P. rosae in carrots and the underlying mechanisms, with special reference to chemical factors. In one we compared only two cultivars using egg-laying traps (details below). In the other four the design was similar to one already reported for use with IOBC cultivars (Ellis and Hardman, 1981). Essentially, there were three blocks each containing one replicate of each cultivar randomized within the block. Block dimensions were 5 x 5 m with 2 m between blocks. All carrot seed was hand-sown in 5-m rows spaced 40 or 50 cm apart within the blocks. Rows were perpendicular to a hedge and began 2 m away from it. Two guard rows terminated the layout of each trial. Carrot seed of the following eight cultivars tested was supplied by the IOBC through the National Vegetable Research Station (NVRS), Wellesbourne, U.K.: Clause's Original Sytan (hereafter Sytan), Long Chantenay, Vertou L.D. (hereafter Vertou), Danvers Half Long 126, Gelbe Rheinische, Clause's Jaune
CARROT ROOT RESISTANCE TO CARROT FLY
1869
Obtuse Du Doubs, St. Valery, Royal Chantenay Elite. They represented a selection of European stocks ranging from susceptible to resistant (Ellis and Hardman, 1981). Caulfield Seed Merchants, Dublin, supplied seed of Regulus Imperial and Chantenay-Red Cored-Elite representing, respectively, the extremes of resistance and susceptibility previously reported from this laboratory (Guerin and Ryan, 1984).
Extent of Resistance Two field experiments assessed the extent of resistance on contrasting soil types. All 10 cultivars were compared on peaty soil, comprising layered fen peat with Menyanthese trifoliata, non-Sphagnum moss and intermittent birch remains over wood fen, at the Peatland Experimental Station, The Agricultural Institute, Lullymore, County Kildare in 1982. The 1983 experiment was made on silty loam soil at University College Dublin, Belfield, using only the eight cultivars as supplied by the IOBC. Both experiments were harvested in late October and the roots graded for damage into five agreed categories of damage (Ellis et al., 1978): 0% (clean), < 5 % , 5-25%, 26-50%, and >50% of root surface damaged. Numbers and total weight of roots in each grade were recorded for each row. The data were subjected to analysis of variance as: number of roots; percentage unattacked roots; percentage of carrots with less than 5% of root surface damaged, i.e., percentage of marketable roots; and root damage index based on a grading system described by Ellis et al. (1978). These grades ranged from one for marketable roots to four for roots with more than 50 % of the surface damaged. To allow for variations between cultivars in root density, the data were subjected to covariance analysis and derived means were compared by the least significant difference (LSD) test.
Mechanisms of Resistance These were investigated by three experiments in 1981, 1982, and 1983, all on the peatland site of Lullymore. Essentially, the population densities of successive life stages were estimated to compare the mortality of each stage between cultivars. Egg Sampling. Egg numbers were estimated on nine and 13 occasions for first and second generations of 1982 and on nine occasions for the 1983 experiment, from each of five plants, selected at random, in each row of the center block. Egg densities per cultivar were estimated from those in soil collected in a spoon (5 cm diameter) from the area surrounding the plant within a radius of 5 cm from the root and to a depth of 2.5 cm as previously described (Guerin and Ryan, 1984). Larval Sampling. Six times during the 1981 experiment and 13 times in 1982, carrots were sampled by taking five random batches of two carrots per
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MAKIAND RYAN
row to give a total of 30 carrot plants for each cultivar (Guerin and Ryan, 1984). In addition to numbers of larvae, observations were made on numbers of attacked roots and mines. A mine is defined as an area of carrot 2 cm in diameter on the surface extending towards the core (Wright and Ashby, 1946). Pupal Sampling. Pupal numbers were assessed on January 12, 1983, by collecting three soil samples per row from each block, or a total of nine samples per cultivar, using a square metal frame (25 x 25 x 15 cm deep). This was pushed into the soil surrounding the plants with the carrots centered in the frame. Then the soil sample was collected by pulling out the frame with the soil, which was poured into a plastic bag. Carrot roots were counted in the laboratory, and pupae were extracted from the soil by flotation using a saturated solution of magnesium sulfate. Pupae were collected with a paint brush, streaked on white filter paper, and counted. The number of roots per frame was not constant, so pupal numbers are expressed per root rather than per area.
Root Factors The 1983 experiment differed from the usual design by concentrating on factors affecting egg numbers on only two cultivars, Vertou and Long Chantenay, representing the extremes of resistance and susceptibility, respectively. First, numbers of first-generation eggs were assessed by the spoon method on nine occasions at three-day intervals, from June 22 to August 16, on four plants selected at random from each plot. Numbers of second-generation eggs were compared by the use of egg traps originally designed for use with cabbage root fly, Delia radicum (brassicae) L., (Freuler and Fischer, 1983). They were made of thin layers of felt material rolled down into a disk-shaped trap (5 cm diam. x 2 cm thick; Figure 1). Eggs are laid on the top surface of the traps between the felt layers. We used the traps in two forms: (A) those with a sponge centerpiece that encircled the stem of the plant on the soil surface such that the root was completely covered, and (B) those without such a centerpiece, thus exposing the top of the root. This comparison tested the hypothesis that manipulating the amount of root exposed, and presumably the quantity of root volatiles released, would affect the numbers of eggs laid. It is generally accepted that larvae feed on the slender side roots before invading the main root (Gorham, 1934; Van't Sant, 1961; Whitcomb, 1938; Beirne, 1971; St/idler, 1971; Jones, 1979; Guerin and Ryan, 1984); Overbeck (1978) reported that one third of larval development occurs in such side roots. We sought to compare the extent to which the first-instar larva of Psila feeds on side roots of resistant and susceptible cultivars before moving on to the main root. Accordingly, the surfaces of side roots in samples of 40 roots each from Vertou and Long Chantenay, taken at three-day intervals on each of seven occasions from August 4 to 19, 1983, were examined microscopically (x40). In
CARROT ROOT RESISTANCE TO CARROT FLY
1871
FIG. ][. Egg-traps used to assess the effect on oviposition of exposing the carrot root top. In A The sponge centerpiece completely covers the root top and in B the absence of the centerpiece exposes it. In both, the foliage is unaffected. With the root top covered, flies did not distinguish between Vertou and Long Chantenay in ovipositing, but when it was exposed significantly more eggs were laid on Long Chantenay as in regular field situations. Clearly, the foliage alone is inadequate to elicit this preference and must be supplemented by input from the root which, in the present comparison, is likely to be chemical.
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MAKI AND RYAN
addition, the cotton-blue lactophenol staining method, used to detect nematodes in plants (Souchey, 1970), was exploited in an effort to identify larvae of P. rosae in 175 side roots taken from each cultivar.
Statistical Analysis All data for the experiments on resistance mechanisms relating to numbers of eggs, larvae, and mines were subjected to a square-root transformation (X = ~ n + 1) followed by analysis of variance; means were compared by the Duncan's multiple-range test (Duncan, 1955). RESULTS
Extent of Resistance The experiments on peat and mineral soil gave essentially similar results. All differences between cultivars reported below are statistically significant (P < 0.05) unless otherwise stated. On peat soil, Vertou had less damage, more unattacked roots, and more marketable roots than Regulus Imperial, St. Valery, and Long Chantenay (Table 1). On mineral soil, Clause's Original Sytan and Vertou had less damage than St. Valery, Danvers Half Long 126, Gelbe Rheinische, Long Chantenay, and Royal Chantenay Elite; more unattacked roots than all the other six cultivars, which did not differ significantly from each other; and more marketable roots than Danvers Half Long 126, St. Valery, Gelbe Rheinische, and Royal Chantenay Elite (Table 2). We consolidated the results from each experiment into a single ranking system by assigning a value of 1 to the least resistant cultivar under each criteflon; values of 10 and 8 were assigned to the most resistant cultivar in peat and mineral experiments, respectively, to take account of the different number of cultivars tested. This allows a possible maximum score of 30 and 24 for each cultivar in the peat and mineral soil experiments, respectively. For comparisons between experiments, cultivars were then rated by expressing their scores as a percentage of the maximum possible score (Tables 1 and 2). On peat soil, Vertou was the most resistant cultivar (100%), followed by Gelbe Rheinische (87 %), and Regulus was the most susceptible (10 %). On mineral soil, the two most resistant cultivars were Sytan (100%) and Vertou (88%), and Danvers Half Long 126 (17%) was the most susceptible.
Mechanisms of Resistance In the 1981 experiment, when observations were confined to the second generation, more larvae and more mines per root were recorded on Long Chantenay than on all the other cultivars, which did not differ significantly from each other in either respect (Table 3).
Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Chantenay-Red Cored-Elite Sytan
Cultivar a ab abc abc
31.0 bcd 29.4 bcd 23.8 cd
35.8 abc
36.7 abc
38.0 abc
48.5 44.5 39.0 38.2
Clean roots (%)c
Chantenay-Red Cored-Elite Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Ctause's Jaune Obtuse Du Doubs Sytan
Cultivar a ab ab abc
43.8 bcd 42.3 bcd 36,3 cd
48.3 abc
49.5 abc
49.6 abc
63.9 63.9 57.5 51.3
Marketable roots (%)J
Chantenay-Red Cored-Elite Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Sytan
Cultivar
aMeans in the same column with different letters are significantly different (P < 0.05). bMean damage scores which range from 1 for marketable roots to 4 for root with more than 50% of the surface damaged. c Unattacked roots, dPercentage of carrots with less than 5% of root surface damaged. eScoring system based by assigning 1 to the least resistant and 10 to the most resistant cultivars with corresponding intervening values.
2.08 bcd 2.08 bcd 2.22 cd
1.99 abc
1.96 abc
1.91 abc
Chantenay-Red Cored-Elite Clause's Jaune Obtuse Du Doubs Danvers Half Long 126 St. Valery Long Chantenay Regulus Imperial
a ab ab abc
1.59 1.68 1.75 1.87
Vertou Royal Chantenay Elite Gelbe Rheinische Sytan
Cultivar
Root damage index b
23 10
27
40
57
60
100 87 83 63
Score as % of max. possible e
TABLE 1. RANKED ORDER UNDER THREE CRITERIA OF CARROT FLY DAMAGE ON TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) IN 1982 WITH CONSOLIDATION INTO SINGLE RANKING TO EXPRESS EXTENT OF RESISTANCE a
Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Gelbe Rheinische St. Valery
Danvers Half Long 126
2.75 c 2.75 c
2.91 c
2.83 c 2.89 c
Sytan Vertou Long Chantenay
1.74 a 2.07 ab 2.67 bc
Cultivar
6.4 b
15.6 b 12.6 b
18.2 b 16.9 b
46.5 a 38.6 a 18.5 b
Clean roots (%)c
Danvers Half Long 126
Gelbe Rheinische St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Long Chantenay Royal Chantenay Elite
Cultivar
18.9 c
23.6 c 22.3 c
29.4 bc 26.8 c
61.1 a 50.2 ab 29.6 bc
Marketable roots (%)d
Danvers Half Long 126
Gelbe Rheinische St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Long Chantenay Royal Chantenay Elite
Cultivar
"Means in the same column with different letters are significantly different (P < 0.05). bMean damage scores, which range from 1 for marketable roots to 4 for root with more than 50% of the surface damaged. c Unattacked roots. dpercentage of carrots with less than 5 % of root surface damaged. ~Scoring system based by assigning 1 to the least resistant and 8 to the most resistant cultivars with corresponding intervening values.
Gelbe Rheinische Danvers Half Long 126 St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Royal Chantenay Elite Long Chantenay
Cultivar
Root damage index b
17
38 21
63 58
100 88 67
Score as % of max. possiblee
TABLE 2. RANKED ORDER UNDER THREE CRITERIA OF CARROT FLY DAMAGE ON EIGHT CARROT CULTIVARS AT BELFIELD (MINERAL SOIL) IN 1983 WITH CONSOLIDATION INTO SINGLE RANKING TO EXPRESS THE EXTENT OF RESISTANCE. a
Danvers Half Long 126 Regulus Imperial Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Long Chantenay
1.09 a
1.10 a
1.11 a
1.11 a
1.19 b
Royal Chantenay Elite
Clause's Jaune Obtuse Du Doubs Regulus Imperial
Long Chantenay
1.19 b
1.13 ab
1.13 ab
1.12 ab
1.11 ab
1.11 ab
1.09 a
1.07 a 1.8 a 1.08 a
No. of mines b
aMeans in the same column with different letters are significantly different (P < 0.05). bData transformed x/~-n+ 1.
St. Valery
Vertou Gelbe Rheinische Chantenay-Red Cored-Elite Sytan
1.08 a
1.08 a
1.05 a 1.06 a 1.07 a
Cultivar
Danvers Half Long 126 Sytan
Vertou Gelbe Rheinische Cbantenay-Red Cored-Elite St. Valery
Cultivar
No. of larvae b
Danvers Half Long 126
Royal Chantenay Elite
Clause's Jaune Obtuse Du Doubs St. Valery
Gelbe Rheinische
Chantenay-Red Cored-Elite Vertou
Sytan Long Chantenay Regulus Imperial
Cultivar
1.03
1.03
1.03
1.02
1.02
1.02
1.01
1.00 1.00 1.01
No. of mines/invaded larva
TABLE 3. RANKED ORDER IN TERMS OF MEAN NUMBERS OF LARVAE AND MINES FOR TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) IN 1981 TO ASSESS MECHANISMS OF RESISTANCE a
k/I
O
O > ~v
~z
0 0
0
>
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MAKI AND RYAN
In the 1982 experiment (first generation), fewer eggs were laid on Vertou, Sytan, and three other cultivars than on Royal Chantenay Elite, Regulus Imperial, and Long Chantenay (Table 4). There was, however, no significant difference between the numbers of larvae subsequently recorded on the roots of these cultivars (Table 4). Vertou, Clause's Jaune Obtuse Du Doubs, and Sytan had fewer mines per root than Long Chantenay, Chantenay-Red Cored-Elite, and Royal Chantenay Elite. The ratio of eggs laid to larvae established in main roots ranged from 7 (Gelbe Rheinische) to 11 (Long Chantenay) (Table 4). Using the scoring system, Vertou was the most resistant (100%) followed by Clause's Jaune Obtuse Du Doubs (90%), St. Velery (77 %), and Sytan (73 %). In contrast, Long Chantenay was the most susceptible (27%). Experimental results from the second generation were consistent with those from the first. Vertou had fewer eggs than Danvers Half Long 126 and Gelbe Rheinische. Furthermore, Vertou had fewer larvae than Gelbe Rheinische, Long Chantenay, Danvers Half Long 126, and Regulus Imperial. The ratio of eggs laid to larvae established in main roots ranged from 10 (Danvers half Long 126) to 12 (Sytan and Vertou) (Table 5). Vertou, Sytan, and three other cultivars had fewer mines per root than Gelbe Rheinische, Long Chantenay, Danvers Half Long 126, and Regulus Imperial (Table 5). Larval damage increased on all cultivars from the middle of September and peaked in November. A comparison between Vertou (resistant) and Long Chantenay (susceptible) showed that consistently more eggs, larvae, mines, and damaged roots were recorded on Long Chantenay than on Vertou. Percentage larval survival to the pupal stage ranged from 66% (St. Valery) to 98% (Vertou). The same scoring system showed Vertou again the most resistant cultivar (82%), followed by Clause's Jaune Obtuse Du Doubs (80 %) and Sytan (78 %). Long Chantenay was the third most susceptible cultivar (38 %). In the 1983 experiment (first generation) fewer (P < 0.001) eggs were laid on Vertou than on Long Chantenay as judged by the flotation method (Table 6). In the second generation, the use of egg trap A (root top enclosed) abolished this difference. But use of egg trap B (root top exposed) restored the significant difference between Vertou and Long Chantenay (Table 6). This unambiguously demonstrates that the root can regulate preference/nonpreference oviposition by the female fly. The mean number of side roots per carrot root of the two cultivars Vertou and Long Chantenay was identical. Lactophenol staining did not detect Psila larvae inside such side roots. However, larval feeding areas were readily identified by examination with a binocular microscope. Each comprised a shallow pit in the epidermis ( < 2 mm long and 50-100 tzm deep), with a rust-colored perimeter. Alternatively this took the form of a narrow scar, more than 2 mm long. However, there was no significant difference between mean numbers of side roots infested on Vertou (1.3) and Long Chantenay (1.2) (N = 7).
1.57 b
Long Chantenay
Regulus Imperial Chantenay-Red Cored-Elite Gelbe Rheinische
Danvers Half Long 126 Royal Chantenay Elite
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Long Chantenay
Cultivar
1.22 a
1.19 a 1.21 a
1.19 a
1.17 a
1.14 a 1.15 a 1.15 a
1.09 a 1.12 a
No. of larvae b
Danvers Half Long 126 Royal Chantenay Elite Chantenay-Red Cored-Elite Long Chantenay
Gelbe Rheinische
Vertou Clause's Jaune Obtuse Du Doubs Sytan St. Valery Regulus Imperial
Cultivar
1.83 d
1.78 d 1.78 d
1.68 cd
1.65 cd
1.42 ab 1.53 abc 1.56 abc
1.36 a 1.41 ab
No. of mines b
Royal Chantenay Elite Chantenay-Red Cored-Elite Long Chantenay
Regulus Imperial
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Danvers Half Long 126 Gelbe Rheinische
Cultivar
aMeans in the same column with different letters are significantly different (P < 0.05). bData transformed ~/n + 1. CScoring system based by assigning 1 to the least resistant and 10 to the most resistant cultivars with corresponding intervening values.
1.55 b 1.57 b
1.40 ab
1.29 ab
1.17 a 1.22 a 1.27 a
1.12 a 1.16 a
Danvers Half Long 126 Royal Chantenay Elite Regulus Imperial
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Chantenay-Red Cored-Elite Gelbe Rheinische
Cultivar
No. of eggsb
27
33 33
37
37
77 73 43
100 90
Score as % of max. possiblec
TABLE 4. RANKED ORDER IN TERMS OF MEAN NUMBERS OF EG~S, LARVAE AND MINES PER ROOT FOR TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) DUPING FIRST GENERATION 1982 WITH CONSOLIDATION INTO SINGLE RANKING TO ASSESS MECHANISMS OF RESISTANCEa
1.72 b
1.61 ab 1.64 b
Long Chantenay Danvers Half Long 126 Regulus Imperial 1.71 c
1.58 bc 1.64 bc
1.46 ab 1.57 bc
1.34 a 1.43 ab
Long Chautenay Danvers Half Long 126 Regulus Imperial
Royal Chantenay Elite Gelbe Rheinische
1.73 d
1.63 bcd 1.66 cd
1.49 abc 1.59 bcd
Clause's Jaune Obtuse 1.45 ab Du Doubs Chantenay-Red 1.45 ab Cored-Elite St. Valery 1.45 ab
Vertou Sytan
Cultivar
No. of mines b
Royal Chantenay Elite Danvers Half Long 126 Regulus Imperial Chantenay-Red Cored-Elite Vertou
88.3
Clause's Jaune Obtuse Du Doubs Sytan
97.9
97.6 97.6
94.2 96.7
91.4
87.9
65.9 84.3
Long Chantenay
St. Valery Gelbe Rheinische
Cultivar
Survival (%, larvae to pupae)
Long Chantenay Danver Half Long 126 Regulus Imperial
Chantenay-Red Cored-Elite Royal Chantenay Elite Gelbe Rheinische
St. Valery
Vertou Clause's Jaune Obtuse Du Doubs Sytan
Cultivar
20
38 28
54 48
56
66
78
82 80
Score as % of max. possible'
AND M I N E S PER R O O T AND PERCENTAGE L A R V A L SURVIVAL TO PUPAL
a M e a n s in the same c o l u m n with different letters are significantly different (P < 0 . 0 5 ) . bData t r a n s f o r m e d x/n + 1. CScoring system b a s e d b y a s s i g n i n g 1 to the least resistant a n d 10 to the m o s t resistant cultivars with c o r r e s p o n d i n g i n t e r v e n i n g values.
Regulus Imperial Danvers Half Long 126 Gelbe Rheinische
1.57 ab 1.59 ab
1.41 ab
Chantenay-Red Cored-Elite Royal Chantenay Elite Gelbe Rheinische
Chantenay-Red Cored-Elite St. Valery Long Chantenay
1.56 ab
Clause's Jaune Obtuse 1.40 ab Du Doubs St. Valery 1.41 ab
1.31 a 1.39 ab
Clause's Jaune Obtuse 1.41 ab Du Doubs Sytan 1.43 ab
Cultivar
No. of larvae b
Vertou Sytan
No. of egss b
Vertou 1.29 a Royal Chantenay Elite 1.40 ab
Cultivar
EGGS,LARVAE
STAGE AT LULLYMORE ( P E A T SOIL) DURING SECOND GENERATION 1 9 8 2 WITH CONSOLIDATION INTO SINGLE RANKING ~'
TABLE 5. RANKED ORDER FOR M E A N NUMBERS OF
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CARROT ROOT RESISTANCE TO CARROT FLY
TABLE 6. COMPARISON OF VERTOUS (RESISTANT) AND LONG CHANTENAY (SuscEPTIBLE) IN EGG NUMBERS USING SOIL SAMPLING DURING FIRST GENERATION AND EGG TRAPS DURING SECOND GENERATION AT LULLYMORE (PEAT SOIL) IN 1983
First generation
Second generation
Carrot cultivar
No. of eggs/root (soil sample)
No. of eggs per trap Aa
No. of eggs per trap B b
Vertou Long Chantenay Significance
1.88 2.54 P < 0.001
i .05 1.07 NS ~
1.03 1.41 P < 0.05
"Trap A was with a sponge centerpiece. bTrap B was without centerpiece. CNot significant.
DISCUSSION
Vertou was consistently resistant: it was the most resistant cultivar on peat and the second most resistant on mineral soil. Sytan's ranking improved from sixth most resistant on peat to first place on mineral soil. At Wellesboume, U.K., more sinuous mines but fewer nibbles were observed on carrots from fen or peaty soil than on those from mineral soil, suggesting that larvae mined more readily in tissues of peat-grown carrots (Ellis et al., 1978). Such a mechanism could contribute to Sytan's elevation to first place on mineral soil in our experiments. Similarly, Long Chantenay was the second most resistant cultivar in 1982 (peat soil) but the fourth resistant cultivar in 1983 (mineral). Overall, our data are quite consistent with the results of similar experiments in five European countries (Ellis and Hardman, 1981). Using the single criterion of percentage undamaged roots, they also rated Sytan and Vertou as the two most resistant cultivars. Their most susceptible cultivar was St. Valery, with Danvers in second place. Excluding Regulus Imperial, which was not considered by them, we found the most susceptible cultivars to be St. Valery and Long Chantenay in 1982 and Danvers and St. Valery in 1983. Thus, we confirm Sytan and Vertou as the most resistant cultivars and St. Valery as among the most susceptible. In regard to mechanisms, highly significant differences in egg numbers were exhibited by different cultivars during the first (P < 0.001) and second (P < 0.01) generations of 1982. Resistant Vertou had 29% fewer eggs than susceptible Long Chantenay in the first generation of 1982. Such a response confirms nonpreference by the female Psila (A hlberg, 1944; Guerin and Ryan, 1984). The experiment with egg-laying traps showed that exposing only the foliage was insufficient to reproduce differential egg numbers found between
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MAKI AND RYAN
Vertou and Long Chantenay; it was essential to also expose the respective root tops. This effect may be assignable to root volatiles, i.e., attractants and/or oviposition stimulants in root scent. Significantly fewer eggs were laid on foliage of resistant Sytan than on that of susceptible Danvers in laboratory experiments (Guerin and St/idler, 1982), which is consistent with the data from our field experiments. However, this preference was abolished when artificial leaves impregnated with leaf surface extracts from the two cultivars were compared in the laboratory. Furthermore, in a comparison of the effect of headspace vapor, Sytan attracted significantly more eggs than Danvers. Hence, the effect of contact with foliage was reproduced by neither extracts nor headspace volatiles of foliage. It was acknowledged that the observations took no account of root odor (Guerin and St/idler, 1982). The present data indicate that input from the root contributes significantly to differential egg laying. The second mechanism confirmed by our field data was resistance by the main root to larval attack as judged by numbers of larvae, mines, pupae, and damaged roots. This is exemplified by a comparison of Vertou with Long Chantenay and Regulus Imperial in the second generation of 1982. Cultivars Long Chantenay and Regulus Imperial had similar egg numbers to Vertou but sustained significantly more larvae, damaged roots, and mines (Table 5). Accordingly, these root effects reflect not only egg numbers but also indicate direct root resistance against the larva. This effect was first reported from a different set of cultivars by Guerin and Ryan (1984). Present data indicate that the number of side roots would not affect root resistance in the two extreme cultivars (Vertou and Long Chantenay) as the mean number of side roots did not differ significantly between the two cultivars. Furthermore, detailed examination of the anatomy of the side roots gave no evidence of differential feeding or differential invasion. Thus, by elimination, root resistance to larval invasion must operate through the main root. There was no evidence of more dead or stunted larvae immediately after establishment in any cultivar, eliminating antibiosis at that time as a mechanism. This suggests that decreased invasion, i.e., nonpreference exerted by the main root, governs larval establishment in such roots. At least five compounds, c~-ionone, ~-ionone, bornyl acetate, biphenyl, and 2,4-dimethyl styrene, attract larvae to roots (Ryan and Guerin, 1982). One compound, trans-2-nonenal, repelled and killed the larvae in laboratory experiments (Guerin and Ryan, 1980). Furthermore, combined concentrations of the attractants, but especially of 2,4-dimethyl styrene, were fivefold greater in the main root of the susceptible Chantenay-Red Cored-Elite as compared with resistant Regulus Imperial (Guerin and Ryan, 1984). Accordingly, it is reasonable to conclude that nonpreference, as influenced by chemical cues, affects root resistance to the larva. Another possible resistance mechanism by the main root is antibiosis
CARROT ROOT RESISTANCE TO CARROT FLY
1881
against the feeding larva, i.e., if after prolonged feeding, larval growth, development, and survival were decreased (Campbell, 1983). In the second generation of 1982, larval survival in St. Valery and Sytan was 32% and 7% less, respectively, than in Vertou (100% survival). So antibiosis is another mechanism by which main root resistance operates, although it does not materially contribute to the resistance of Vertou and Sytan. Antibiosis has previously been assigned to carrot on the basis of differential invasion of roots from inoculation with equal numbers of eggs (Guerin et al., 1981). However, the result did not preclude differential invasion due to nonpreference behavior by the larvae in response to root chemicals. The present data provide an unambiguous demonstration of antibiosis. As this mechanism takes effect through physiological inhibitors, toxins, and decreased nutrient levels (Beck, 1965), it is reasonable to implicate root chemicals in its action against the Psila larva. In summary, the present data identify root-mediated resistance as operating by: (1) eliciting fewer eggs (nonpreference by the female), (2) independently decreasing larval invasion (nonpreference by the larvae), and (3) antibiosis against the root-established larvae. Accordingly, root-mediated resistance has a pervasive role in carrot resistance to Psila. Root chemicals are already established as contributing to the second of these mechanisms (Guerin and Ryan, 1984), but it is now apparent that they also contribute to both the former and the latter. Accordingly, main root chemical constituents may be crucial in carrot resistance to Psila. Acknowledgments--We thank Dr. P.R. Ellis, National Vegetable Research Station, Wellesbourne, U.K., for supplying seed and some statistical analyses.
REFERENCES ~xHLBERG, O. 1944. Olika morotsorter och morotflugan. Vaextskyddsnotiser. 8:49-50. BECK, S.D. 1965. Resistance of plants to insects. Annu. Rev. Entomol. 10:207-232. BEIRNE, B.P. 1971. Pest insects of annual crop plants in Canada: Psila rosae (F.), the carrot rust fly. Mere. Entomol. Soc. Can. 78:63-65. CAMeBELL, C.A.M. 1983. Antibiosis in hop (Humulus lupulus) to the damson-hop aphid, Phorodon humuli. EntomoL Exp. Appl. 33:57-62. DUNCAN, D.B. 1955. Multiple range and multiple F test. Biometrics 11:1-42. ELLIS, P.R., and HARDMAN, J.A. 1981. The consistency of the resistance of eight carrot cultivars to carrot fly attach at several centres in Europe. Ann. Appl. Biol. 98:491-497. ELLIS, P.R., WHEATLEY, G.A., and HARDMAN,J.A. 1978. Preliminary studies of carrot susceptibility to carrot fly attack. Ann. Appl. Biol. 88:159-170. FREULER, J., and FISCHER, S. 1983. Le Piege a Oeufs, nouveaux moyen de prevision d'attague pour la mouche du chou, Delia radicum (brassicae) L. Rev. Suisse Vitie. Arboric. Hortic. 15:107-110.
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GORHAM, R.P. 1934. Control of carrot mst fly, Psila rosae Fab. Rep. Que. Soc. Prot. Plants 26:90-96. GUERIN, P.M., and RVAN, M.F. 1980. Insecticidal effect of trans-2-nonenal, a constituent of carrot root. Experientia 36:1387-1388. GUERIN, P.M., and RYAN, M.F. 1984. Relationship between root volatiles of some carrot cultivars and their resistance to the carrot fly, Psila rosae. Entomol. Exp. Appl. 36:217-224. GUERIN, P.M., and ST.~DLER, E. 1982. Host odour perception in three phytophagous Diptera--a comparative study, pp. 95-105, in J.H. Visser and A.K. Minks (eds.). Proceedings of the 5th International Symposium on Insect-Plant Relationships. Pudoc, Wageningen. GUERIN,P.M., GFELLER,F., and ST,~DLER,E. 1981. Carrot resistance to the carrot fly--Contributing Factors. Report o f the Second Meeting, Eucarpia/IOBC Working Group Breeding for Resistance to Insects and Mites. West Pal. Reg. Sect. Bull. IV:63-65. JONES, O.T. 1979. The responses of carrot fly larvae, Psila rosae, to compounds of their physical environment. Ecol. Entomol. 4:327-334. OVERBECK,H. 1978. Untersuchnngen zum Eiblage- und Befallsuerholten der Mohrenfiiege, Psila rosae F. (Diptera: Psilidae), im Hinblick auf eine modifizierte chemische Bekampfung. Mitt. Biol. Bundesanstalt Berlin-Dahlem Heft. 183:1-183. RYAN, M.F., and GUERIN, P.M. 1982. Behavioural responses of the carrot fly larva, Psila rosae, to carrot root volatiles. Physiol. Entomol. 7:315-324. RYAN, M.F., GUERIN, P.M., and BEHAN, M. 1978. Possible roles for naturally occurring chemicals in the biological control of the carrot fly, pp. 130-153, in J.J. Duggan (ed.). Biological Control. Symposium of the Royal Irish Academy. SOUCHEY,J.F. 1970. Laboratory methods for work with plant and soil nematodes. Technical Bulletin (No. 2). M.A.F.F., HMSO, London. STADLER, E. 1971. Uber die Orientierung und das Wirtswahlverhalten der Mohrenfliege, Psila rosae F. (Diptera:Psilidae) I. Larven. Z. Angew. Entomol. 69:425-438. VAN'T, SANT, L.E. 1961. Levenswijze en bestrijding van de wortelvlieg (Psila rosae F.) in Nededand. Versl. Landbouwk. Onderz. 67:1-131. WHITCOMB,W.D. 1938. The carrot rest fly. Bull. Mass. Agric. Exp. Stn. 352:1-36. WRIGHT, D.W., and ASHBY, D.G. 1946. Bionomics of carrot fly. I. The infestation and sampling of carrot crops. Ann. Appl. Biol. 33:69-77.
ROOT-MEDIATED EFFECTS IN CARROT RESISTANCE TO THE CARROT FLY, P s i l a r o s a e
AMEL
M A K I and M . F . R Y A N Department of Zoology University College Dublin BeIfield, Dublin 4, Ireland
(Received March 21, 1988; accepted September 20, 1988) Abstract--Field experiments on two different soil types in Ireland assessed the extent and mechanisms of resistance to Psila rosae (F.), the carrot fly, with emphasis on the role of the carrot root. Ten carrot cultivars gave consistent results in terms of resistant and susceptible cultivars. Nonpreference oviposition was confirmed as a mechanism, and the use of egg traps, providing differential exposure of the main root, showed this was regulated by root factors, probably chemical constituents. Independent main root resistance to the larva was also confirmed, and this effect was established as consistent with a chemically mediated nonpreference. Antibiosis by the root was demonstrated. Such effects in three different modes indicate that main root properties are crucial in carrot resistance to Psila and suggest a pervasive influence of root chemicals on such resistance. Key Words--Carrot resistance, carrot fly, Psila rosae, Diptera, Psilidae, main root factors, nonpreference oviposition, root resistance to larvae, antibiosis, root chemicals.
INTRODUCTION T h e first report o f resistance in carrot (Daucus carota L.) against the carrot fly [Psila rosae (F.)] indicated n o n p r e f e r e n c e in e g g - l a y i n g as a contributing factor ( A h l b e r g , 1944). M o s t subsequent investigations e m p h a s i z e d screening for resistance, w h i c h led to the f o r m a t i o n in 1976 o f a w o r k i n g group o f the International O r g a n i s a t i o n for B i o l o g i c a l C o n t r o l ( I O B C ) with representatives f r o m nine E u r o p e a n countries. T h e i r data f r o m 12 sites demonstrated resistance as h a v i n g sufficient effect and c o n s i s t e n c y to suggest a role for resistant cultivars in the integrated control o f Psila (Ellis and H a r d m a n , 1981); h o w e v e r , the 1867 0098-0331/89/0600-1867506.00/0 9 1989 Plenum Publishing Corporation
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mechanisms of resistance were not ascertained. These were examined in this laboratory, where we also addressed the role of chemical factors using a range of cultivars different from those of the IOBC (Ryan et al., 1978). Our field experiments confirmed nonpreference at the level of oviposition and detected a new effect in the form of root resistance to the larva that was independent of resistance to egg-laying (Guerin and Ryan, 1984); that is, two cultivars attracting equally few eggs could sustain widely different degrees of root damage. As such damage is the direct cause of crop losses, it follows that root resistance to the larva is a fundamental requirement in breeding resistance (Guerin and Ryan, 1984). It was not possible to decide if this operated through larval nonpreference or antibiosis, and this question is addressed in the present report. It was also clear that intact roots of resistant varieties released substantially less volatile material than susceptible ones as judged by comparison of steam distillates and of headspace vapors entrapped by Porapak Q (Guerin and Ryan, 1984). We now report on field experiments with the IOBC range of cultivars. The results confirm nonpreference at the level of oviposition, independent root resistance to the larva, and show, in addition, that the latter is consistent with a chemically mediated nonpreference effect. The results unambiguously demonstrate antibiosis as independently elicited by the root, which also involves chemicals. Finally, and perhaps surprisingly, they indicate that root factors, as distinct from foliage ones, can govern nonpreference oviposition. Collectively, such data establish the root as capable of regulating carrot resistance to Psila, probably through its chemical constituents.
METHODS AND MATERIALS
A total of five field experiments were conducted over three years to assess both the extent of resistance to P. rosae in carrots and the underlying mechanisms, with special reference to chemical factors. In one we compared only two cultivars using egg-laying traps (details below). In the other four the design was similar to one already reported for use with IOBC cultivars (Ellis and Hardman, 1981). Essentially, there were three blocks each containing one replicate of each cultivar randomized within the block. Block dimensions were 5 x 5 m with 2 m between blocks. All carrot seed was hand-sown in 5-m rows spaced 40 or 50 cm apart within the blocks. Rows were perpendicular to a hedge and began 2 m away from it. Two guard rows terminated the layout of each trial. Carrot seed of the following eight cultivars tested was supplied by the IOBC through the National Vegetable Research Station (NVRS), Wellesbourne, U.K.: Clause's Original Sytan (hereafter Sytan), Long Chantenay, Vertou L.D. (hereafter Vertou), Danvers Half Long 126, Gelbe Rheinische, Clause's Jaune
CARROT ROOT RESISTANCE TO CARROT FLY
1869
Obtuse Du Doubs, St. Valery, Royal Chantenay Elite. They represented a selection of European stocks ranging from susceptible to resistant (Ellis and Hardman, 1981). Caulfield Seed Merchants, Dublin, supplied seed of Regulus Imperial and Chantenay-Red Cored-Elite representing, respectively, the extremes of resistance and susceptibility previously reported from this laboratory (Guerin and Ryan, 1984).
Extent of Resistance Two field experiments assessed the extent of resistance on contrasting soil types. All 10 cultivars were compared on peaty soil, comprising layered fen peat with Menyanthese trifoliata, non-Sphagnum moss and intermittent birch remains over wood fen, at the Peatland Experimental Station, The Agricultural Institute, Lullymore, County Kildare in 1982. The 1983 experiment was made on silty loam soil at University College Dublin, Belfield, using only the eight cultivars as supplied by the IOBC. Both experiments were harvested in late October and the roots graded for damage into five agreed categories of damage (Ellis et al., 1978): 0% (clean), < 5 % , 5-25%, 26-50%, and >50% of root surface damaged. Numbers and total weight of roots in each grade were recorded for each row. The data were subjected to analysis of variance as: number of roots; percentage unattacked roots; percentage of carrots with less than 5% of root surface damaged, i.e., percentage of marketable roots; and root damage index based on a grading system described by Ellis et al. (1978). These grades ranged from one for marketable roots to four for roots with more than 50 % of the surface damaged. To allow for variations between cultivars in root density, the data were subjected to covariance analysis and derived means were compared by the least significant difference (LSD) test.
Mechanisms of Resistance These were investigated by three experiments in 1981, 1982, and 1983, all on the peatland site of Lullymore. Essentially, the population densities of successive life stages were estimated to compare the mortality of each stage between cultivars. Egg Sampling. Egg numbers were estimated on nine and 13 occasions for first and second generations of 1982 and on nine occasions for the 1983 experiment, from each of five plants, selected at random, in each row of the center block. Egg densities per cultivar were estimated from those in soil collected in a spoon (5 cm diameter) from the area surrounding the plant within a radius of 5 cm from the root and to a depth of 2.5 cm as previously described (Guerin and Ryan, 1984). Larval Sampling. Six times during the 1981 experiment and 13 times in 1982, carrots were sampled by taking five random batches of two carrots per
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row to give a total of 30 carrot plants for each cultivar (Guerin and Ryan, 1984). In addition to numbers of larvae, observations were made on numbers of attacked roots and mines. A mine is defined as an area of carrot 2 cm in diameter on the surface extending towards the core (Wright and Ashby, 1946). Pupal Sampling. Pupal numbers were assessed on January 12, 1983, by collecting three soil samples per row from each block, or a total of nine samples per cultivar, using a square metal frame (25 x 25 x 15 cm deep). This was pushed into the soil surrounding the plants with the carrots centered in the frame. Then the soil sample was collected by pulling out the frame with the soil, which was poured into a plastic bag. Carrot roots were counted in the laboratory, and pupae were extracted from the soil by flotation using a saturated solution of magnesium sulfate. Pupae were collected with a paint brush, streaked on white filter paper, and counted. The number of roots per frame was not constant, so pupal numbers are expressed per root rather than per area.
Root Factors The 1983 experiment differed from the usual design by concentrating on factors affecting egg numbers on only two cultivars, Vertou and Long Chantenay, representing the extremes of resistance and susceptibility, respectively. First, numbers of first-generation eggs were assessed by the spoon method on nine occasions at three-day intervals, from June 22 to August 16, on four plants selected at random from each plot. Numbers of second-generation eggs were compared by the use of egg traps originally designed for use with cabbage root fly, Delia radicum (brassicae) L., (Freuler and Fischer, 1983). They were made of thin layers of felt material rolled down into a disk-shaped trap (5 cm diam. x 2 cm thick; Figure 1). Eggs are laid on the top surface of the traps between the felt layers. We used the traps in two forms: (A) those with a sponge centerpiece that encircled the stem of the plant on the soil surface such that the root was completely covered, and (B) those without such a centerpiece, thus exposing the top of the root. This comparison tested the hypothesis that manipulating the amount of root exposed, and presumably the quantity of root volatiles released, would affect the numbers of eggs laid. It is generally accepted that larvae feed on the slender side roots before invading the main root (Gorham, 1934; Van't Sant, 1961; Whitcomb, 1938; Beirne, 1971; St/idler, 1971; Jones, 1979; Guerin and Ryan, 1984); Overbeck (1978) reported that one third of larval development occurs in such side roots. We sought to compare the extent to which the first-instar larva of Psila feeds on side roots of resistant and susceptible cultivars before moving on to the main root. Accordingly, the surfaces of side roots in samples of 40 roots each from Vertou and Long Chantenay, taken at three-day intervals on each of seven occasions from August 4 to 19, 1983, were examined microscopically (x40). In
CARROT ROOT RESISTANCE TO CARROT FLY
1871
FIG. ][. Egg-traps used to assess the effect on oviposition of exposing the carrot root top. In A The sponge centerpiece completely covers the root top and in B the absence of the centerpiece exposes it. In both, the foliage is unaffected. With the root top covered, flies did not distinguish between Vertou and Long Chantenay in ovipositing, but when it was exposed significantly more eggs were laid on Long Chantenay as in regular field situations. Clearly, the foliage alone is inadequate to elicit this preference and must be supplemented by input from the root which, in the present comparison, is likely to be chemical.
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addition, the cotton-blue lactophenol staining method, used to detect nematodes in plants (Souchey, 1970), was exploited in an effort to identify larvae of P. rosae in 175 side roots taken from each cultivar.
Statistical Analysis All data for the experiments on resistance mechanisms relating to numbers of eggs, larvae, and mines were subjected to a square-root transformation (X = ~ n + 1) followed by analysis of variance; means were compared by the Duncan's multiple-range test (Duncan, 1955). RESULTS
Extent of Resistance The experiments on peat and mineral soil gave essentially similar results. All differences between cultivars reported below are statistically significant (P < 0.05) unless otherwise stated. On peat soil, Vertou had less damage, more unattacked roots, and more marketable roots than Regulus Imperial, St. Valery, and Long Chantenay (Table 1). On mineral soil, Clause's Original Sytan and Vertou had less damage than St. Valery, Danvers Half Long 126, Gelbe Rheinische, Long Chantenay, and Royal Chantenay Elite; more unattacked roots than all the other six cultivars, which did not differ significantly from each other; and more marketable roots than Danvers Half Long 126, St. Valery, Gelbe Rheinische, and Royal Chantenay Elite (Table 2). We consolidated the results from each experiment into a single ranking system by assigning a value of 1 to the least resistant cultivar under each criteflon; values of 10 and 8 were assigned to the most resistant cultivar in peat and mineral experiments, respectively, to take account of the different number of cultivars tested. This allows a possible maximum score of 30 and 24 for each cultivar in the peat and mineral soil experiments, respectively. For comparisons between experiments, cultivars were then rated by expressing their scores as a percentage of the maximum possible score (Tables 1 and 2). On peat soil, Vertou was the most resistant cultivar (100%), followed by Gelbe Rheinische (87 %), and Regulus was the most susceptible (10 %). On mineral soil, the two most resistant cultivars were Sytan (100%) and Vertou (88%), and Danvers Half Long 126 (17%) was the most susceptible.
Mechanisms of Resistance In the 1981 experiment, when observations were confined to the second generation, more larvae and more mines per root were recorded on Long Chantenay than on all the other cultivars, which did not differ significantly from each other in either respect (Table 3).
Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Chantenay-Red Cored-Elite Sytan
Cultivar a ab abc abc
31.0 bcd 29.4 bcd 23.8 cd
35.8 abc
36.7 abc
38.0 abc
48.5 44.5 39.0 38.2
Clean roots (%)c
Chantenay-Red Cored-Elite Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Ctause's Jaune Obtuse Du Doubs Sytan
Cultivar a ab ab abc
43.8 bcd 42.3 bcd 36,3 cd
48.3 abc
49.5 abc
49.6 abc
63.9 63.9 57.5 51.3
Marketable roots (%)J
Chantenay-Red Cored-Elite Danvers Half Long 126 Long Chantenay St. Valery Regulus Imperial
Vertou Gelbe Rheinische Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Sytan
Cultivar
aMeans in the same column with different letters are significantly different (P < 0.05). bMean damage scores which range from 1 for marketable roots to 4 for root with more than 50% of the surface damaged. c Unattacked roots, dPercentage of carrots with less than 5% of root surface damaged. eScoring system based by assigning 1 to the least resistant and 10 to the most resistant cultivars with corresponding intervening values.
2.08 bcd 2.08 bcd 2.22 cd
1.99 abc
1.96 abc
1.91 abc
Chantenay-Red Cored-Elite Clause's Jaune Obtuse Du Doubs Danvers Half Long 126 St. Valery Long Chantenay Regulus Imperial
a ab ab abc
1.59 1.68 1.75 1.87
Vertou Royal Chantenay Elite Gelbe Rheinische Sytan
Cultivar
Root damage index b
23 10
27
40
57
60
100 87 83 63
Score as % of max. possible e
TABLE 1. RANKED ORDER UNDER THREE CRITERIA OF CARROT FLY DAMAGE ON TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) IN 1982 WITH CONSOLIDATION INTO SINGLE RANKING TO EXPRESS EXTENT OF RESISTANCE a
Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Gelbe Rheinische St. Valery
Danvers Half Long 126
2.75 c 2.75 c
2.91 c
2.83 c 2.89 c
Sytan Vertou Long Chantenay
1.74 a 2.07 ab 2.67 bc
Cultivar
6.4 b
15.6 b 12.6 b
18.2 b 16.9 b
46.5 a 38.6 a 18.5 b
Clean roots (%)c
Danvers Half Long 126
Gelbe Rheinische St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Long Chantenay Royal Chantenay Elite
Cultivar
18.9 c
23.6 c 22.3 c
29.4 bc 26.8 c
61.1 a 50.2 ab 29.6 bc
Marketable roots (%)d
Danvers Half Long 126
Gelbe Rheinische St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Long Chantenay Royal Chantenay Elite
Cultivar
"Means in the same column with different letters are significantly different (P < 0.05). bMean damage scores, which range from 1 for marketable roots to 4 for root with more than 50% of the surface damaged. c Unattacked roots. dpercentage of carrots with less than 5 % of root surface damaged. ~Scoring system based by assigning 1 to the least resistant and 8 to the most resistant cultivars with corresponding intervening values.
Gelbe Rheinische Danvers Half Long 126 St. Valery
Sytan Vertou Clause's Jaune Obtuse Du Doubs Royal Chantenay Elite Long Chantenay
Cultivar
Root damage index b
17
38 21
63 58
100 88 67
Score as % of max. possiblee
TABLE 2. RANKED ORDER UNDER THREE CRITERIA OF CARROT FLY DAMAGE ON EIGHT CARROT CULTIVARS AT BELFIELD (MINERAL SOIL) IN 1983 WITH CONSOLIDATION INTO SINGLE RANKING TO EXPRESS THE EXTENT OF RESISTANCE. a
Danvers Half Long 126 Regulus Imperial Royal Chantenay Elite Clause's Jaune Obtuse Du Doubs Long Chantenay
1.09 a
1.10 a
1.11 a
1.11 a
1.19 b
Royal Chantenay Elite
Clause's Jaune Obtuse Du Doubs Regulus Imperial
Long Chantenay
1.19 b
1.13 ab
1.13 ab
1.12 ab
1.11 ab
1.11 ab
1.09 a
1.07 a 1.8 a 1.08 a
No. of mines b
aMeans in the same column with different letters are significantly different (P < 0.05). bData transformed x/~-n+ 1.
St. Valery
Vertou Gelbe Rheinische Chantenay-Red Cored-Elite Sytan
1.08 a
1.08 a
1.05 a 1.06 a 1.07 a
Cultivar
Danvers Half Long 126 Sytan
Vertou Gelbe Rheinische Cbantenay-Red Cored-Elite St. Valery
Cultivar
No. of larvae b
Danvers Half Long 126
Royal Chantenay Elite
Clause's Jaune Obtuse Du Doubs St. Valery
Gelbe Rheinische
Chantenay-Red Cored-Elite Vertou
Sytan Long Chantenay Regulus Imperial
Cultivar
1.03
1.03
1.03
1.02
1.02
1.02
1.01
1.00 1.00 1.01
No. of mines/invaded larva
TABLE 3. RANKED ORDER IN TERMS OF MEAN NUMBERS OF LARVAE AND MINES FOR TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) IN 1981 TO ASSESS MECHANISMS OF RESISTANCE a
k/I
O
O > ~v
~z
0 0
0
>
1876
MAKI AND RYAN
In the 1982 experiment (first generation), fewer eggs were laid on Vertou, Sytan, and three other cultivars than on Royal Chantenay Elite, Regulus Imperial, and Long Chantenay (Table 4). There was, however, no significant difference between the numbers of larvae subsequently recorded on the roots of these cultivars (Table 4). Vertou, Clause's Jaune Obtuse Du Doubs, and Sytan had fewer mines per root than Long Chantenay, Chantenay-Red Cored-Elite, and Royal Chantenay Elite. The ratio of eggs laid to larvae established in main roots ranged from 7 (Gelbe Rheinische) to 11 (Long Chantenay) (Table 4). Using the scoring system, Vertou was the most resistant (100%) followed by Clause's Jaune Obtuse Du Doubs (90%), St. Velery (77 %), and Sytan (73 %). In contrast, Long Chantenay was the most susceptible (27%). Experimental results from the second generation were consistent with those from the first. Vertou had fewer eggs than Danvers Half Long 126 and Gelbe Rheinische. Furthermore, Vertou had fewer larvae than Gelbe Rheinische, Long Chantenay, Danvers Half Long 126, and Regulus Imperial. The ratio of eggs laid to larvae established in main roots ranged from 10 (Danvers half Long 126) to 12 (Sytan and Vertou) (Table 5). Vertou, Sytan, and three other cultivars had fewer mines per root than Gelbe Rheinische, Long Chantenay, Danvers Half Long 126, and Regulus Imperial (Table 5). Larval damage increased on all cultivars from the middle of September and peaked in November. A comparison between Vertou (resistant) and Long Chantenay (susceptible) showed that consistently more eggs, larvae, mines, and damaged roots were recorded on Long Chantenay than on Vertou. Percentage larval survival to the pupal stage ranged from 66% (St. Valery) to 98% (Vertou). The same scoring system showed Vertou again the most resistant cultivar (82%), followed by Clause's Jaune Obtuse Du Doubs (80 %) and Sytan (78 %). Long Chantenay was the third most susceptible cultivar (38 %). In the 1983 experiment (first generation) fewer (P < 0.001) eggs were laid on Vertou than on Long Chantenay as judged by the flotation method (Table 6). In the second generation, the use of egg trap A (root top enclosed) abolished this difference. But use of egg trap B (root top exposed) restored the significant difference between Vertou and Long Chantenay (Table 6). This unambiguously demonstrates that the root can regulate preference/nonpreference oviposition by the female fly. The mean number of side roots per carrot root of the two cultivars Vertou and Long Chantenay was identical. Lactophenol staining did not detect Psila larvae inside such side roots. However, larval feeding areas were readily identified by examination with a binocular microscope. Each comprised a shallow pit in the epidermis ( < 2 mm long and 50-100 tzm deep), with a rust-colored perimeter. Alternatively this took the form of a narrow scar, more than 2 mm long. However, there was no significant difference between mean numbers of side roots infested on Vertou (1.3) and Long Chantenay (1.2) (N = 7).
1.57 b
Long Chantenay
Regulus Imperial Chantenay-Red Cored-Elite Gelbe Rheinische
Danvers Half Long 126 Royal Chantenay Elite
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Long Chantenay
Cultivar
1.22 a
1.19 a 1.21 a
1.19 a
1.17 a
1.14 a 1.15 a 1.15 a
1.09 a 1.12 a
No. of larvae b
Danvers Half Long 126 Royal Chantenay Elite Chantenay-Red Cored-Elite Long Chantenay
Gelbe Rheinische
Vertou Clause's Jaune Obtuse Du Doubs Sytan St. Valery Regulus Imperial
Cultivar
1.83 d
1.78 d 1.78 d
1.68 cd
1.65 cd
1.42 ab 1.53 abc 1.56 abc
1.36 a 1.41 ab
No. of mines b
Royal Chantenay Elite Chantenay-Red Cored-Elite Long Chantenay
Regulus Imperial
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Danvers Half Long 126 Gelbe Rheinische
Cultivar
aMeans in the same column with different letters are significantly different (P < 0.05). bData transformed ~/n + 1. CScoring system based by assigning 1 to the least resistant and 10 to the most resistant cultivars with corresponding intervening values.
1.55 b 1.57 b
1.40 ab
1.29 ab
1.17 a 1.22 a 1.27 a
1.12 a 1.16 a
Danvers Half Long 126 Royal Chantenay Elite Regulus Imperial
Vertou Clause's Jaune Obtuse Du Doubs St. Valery Sytan Chantenay-Red Cored-Elite Gelbe Rheinische
Cultivar
No. of eggsb
27
33 33
37
37
77 73 43
100 90
Score as % of max. possiblec
TABLE 4. RANKED ORDER IN TERMS OF MEAN NUMBERS OF EG~S, LARVAE AND MINES PER ROOT FOR TEN CARROT CULTIVARS AT LULLYMORE (PEAT SOIL) DUPING FIRST GENERATION 1982 WITH CONSOLIDATION INTO SINGLE RANKING TO ASSESS MECHANISMS OF RESISTANCEa
1.72 b
1.61 ab 1.64 b
Long Chantenay Danvers Half Long 126 Regulus Imperial 1.71 c
1.58 bc 1.64 bc
1.46 ab 1.57 bc
1.34 a 1.43 ab
Long Chautenay Danvers Half Long 126 Regulus Imperial
Royal Chantenay Elite Gelbe Rheinische
1.73 d
1.63 bcd 1.66 cd
1.49 abc 1.59 bcd
Clause's Jaune Obtuse 1.45 ab Du Doubs Chantenay-Red 1.45 ab Cored-Elite St. Valery 1.45 ab
Vertou Sytan
Cultivar
No. of mines b
Royal Chantenay Elite Danvers Half Long 126 Regulus Imperial Chantenay-Red Cored-Elite Vertou
88.3
Clause's Jaune Obtuse Du Doubs Sytan
97.9
97.6 97.6
94.2 96.7
91.4
87.9
65.9 84.3
Long Chantenay
St. Valery Gelbe Rheinische
Cultivar
Survival (%, larvae to pupae)
Long Chantenay Danver Half Long 126 Regulus Imperial
Chantenay-Red Cored-Elite Royal Chantenay Elite Gelbe Rheinische
St. Valery
Vertou Clause's Jaune Obtuse Du Doubs Sytan
Cultivar
20
38 28
54 48
56
66
78
82 80
Score as % of max. possible'
AND M I N E S PER R O O T AND PERCENTAGE L A R V A L SURVIVAL TO PUPAL
a M e a n s in the same c o l u m n with different letters are significantly different (P < 0 . 0 5 ) . bData t r a n s f o r m e d x/n + 1. CScoring system b a s e d b y a s s i g n i n g 1 to the least resistant a n d 10 to the m o s t resistant cultivars with c o r r e s p o n d i n g i n t e r v e n i n g values.
Regulus Imperial Danvers Half Long 126 Gelbe Rheinische
1.57 ab 1.59 ab
1.41 ab
Chantenay-Red Cored-Elite Royal Chantenay Elite Gelbe Rheinische
Chantenay-Red Cored-Elite St. Valery Long Chantenay
1.56 ab
Clause's Jaune Obtuse 1.40 ab Du Doubs St. Valery 1.41 ab
1.31 a 1.39 ab
Clause's Jaune Obtuse 1.41 ab Du Doubs Sytan 1.43 ab
Cultivar
No. of larvae b
Vertou Sytan
No. of egss b
Vertou 1.29 a Royal Chantenay Elite 1.40 ab
Cultivar
EGGS,LARVAE
STAGE AT LULLYMORE ( P E A T SOIL) DURING SECOND GENERATION 1 9 8 2 WITH CONSOLIDATION INTO SINGLE RANKING ~'
TABLE 5. RANKED ORDER FOR M E A N NUMBERS OF
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CARROT ROOT RESISTANCE TO CARROT FLY
TABLE 6. COMPARISON OF VERTOUS (RESISTANT) AND LONG CHANTENAY (SuscEPTIBLE) IN EGG NUMBERS USING SOIL SAMPLING DURING FIRST GENERATION AND EGG TRAPS DURING SECOND GENERATION AT LULLYMORE (PEAT SOIL) IN 1983
First generation
Second generation
Carrot cultivar
No. of eggs/root (soil sample)
No. of eggs per trap Aa
No. of eggs per trap B b
Vertou Long Chantenay Significance
1.88 2.54 P < 0.001
i .05 1.07 NS ~
1.03 1.41 P < 0.05
"Trap A was with a sponge centerpiece. bTrap B was without centerpiece. CNot significant.
DISCUSSION
Vertou was consistently resistant: it was the most resistant cultivar on peat and the second most resistant on mineral soil. Sytan's ranking improved from sixth most resistant on peat to first place on mineral soil. At Wellesboume, U.K., more sinuous mines but fewer nibbles were observed on carrots from fen or peaty soil than on those from mineral soil, suggesting that larvae mined more readily in tissues of peat-grown carrots (Ellis et al., 1978). Such a mechanism could contribute to Sytan's elevation to first place on mineral soil in our experiments. Similarly, Long Chantenay was the second most resistant cultivar in 1982 (peat soil) but the fourth resistant cultivar in 1983 (mineral). Overall, our data are quite consistent with the results of similar experiments in five European countries (Ellis and Hardman, 1981). Using the single criterion of percentage undamaged roots, they also rated Sytan and Vertou as the two most resistant cultivars. Their most susceptible cultivar was St. Valery, with Danvers in second place. Excluding Regulus Imperial, which was not considered by them, we found the most susceptible cultivars to be St. Valery and Long Chantenay in 1982 and Danvers and St. Valery in 1983. Thus, we confirm Sytan and Vertou as the most resistant cultivars and St. Valery as among the most susceptible. In regard to mechanisms, highly significant differences in egg numbers were exhibited by different cultivars during the first (P < 0.001) and second (P < 0.01) generations of 1982. Resistant Vertou had 29% fewer eggs than susceptible Long Chantenay in the first generation of 1982. Such a response confirms nonpreference by the female Psila (A hlberg, 1944; Guerin and Ryan, 1984). The experiment with egg-laying traps showed that exposing only the foliage was insufficient to reproduce differential egg numbers found between
1880
MAKI AND RYAN
Vertou and Long Chantenay; it was essential to also expose the respective root tops. This effect may be assignable to root volatiles, i.e., attractants and/or oviposition stimulants in root scent. Significantly fewer eggs were laid on foliage of resistant Sytan than on that of susceptible Danvers in laboratory experiments (Guerin and St/idler, 1982), which is consistent with the data from our field experiments. However, this preference was abolished when artificial leaves impregnated with leaf surface extracts from the two cultivars were compared in the laboratory. Furthermore, in a comparison of the effect of headspace vapor, Sytan attracted significantly more eggs than Danvers. Hence, the effect of contact with foliage was reproduced by neither extracts nor headspace volatiles of foliage. It was acknowledged that the observations took no account of root odor (Guerin and St/idler, 1982). The present data indicate that input from the root contributes significantly to differential egg laying. The second mechanism confirmed by our field data was resistance by the main root to larval attack as judged by numbers of larvae, mines, pupae, and damaged roots. This is exemplified by a comparison of Vertou with Long Chantenay and Regulus Imperial in the second generation of 1982. Cultivars Long Chantenay and Regulus Imperial had similar egg numbers to Vertou but sustained significantly more larvae, damaged roots, and mines (Table 5). Accordingly, these root effects reflect not only egg numbers but also indicate direct root resistance against the larva. This effect was first reported from a different set of cultivars by Guerin and Ryan (1984). Present data indicate that the number of side roots would not affect root resistance in the two extreme cultivars (Vertou and Long Chantenay) as the mean number of side roots did not differ significantly between the two cultivars. Furthermore, detailed examination of the anatomy of the side roots gave no evidence of differential feeding or differential invasion. Thus, by elimination, root resistance to larval invasion must operate through the main root. There was no evidence of more dead or stunted larvae immediately after establishment in any cultivar, eliminating antibiosis at that time as a mechanism. This suggests that decreased invasion, i.e., nonpreference exerted by the main root, governs larval establishment in such roots. At least five compounds, c~-ionone, ~-ionone, bornyl acetate, biphenyl, and 2,4-dimethyl styrene, attract larvae to roots (Ryan and Guerin, 1982). One compound, trans-2-nonenal, repelled and killed the larvae in laboratory experiments (Guerin and Ryan, 1980). Furthermore, combined concentrations of the attractants, but especially of 2,4-dimethyl styrene, were fivefold greater in the main root of the susceptible Chantenay-Red Cored-Elite as compared with resistant Regulus Imperial (Guerin and Ryan, 1984). Accordingly, it is reasonable to conclude that nonpreference, as influenced by chemical cues, affects root resistance to the larva. Another possible resistance mechanism by the main root is antibiosis
CARROT ROOT RESISTANCE TO CARROT FLY
1881
against the feeding larva, i.e., if after prolonged feeding, larval growth, development, and survival were decreased (Campbell, 1983). In the second generation of 1982, larval survival in St. Valery and Sytan was 32% and 7% less, respectively, than in Vertou (100% survival). So antibiosis is another mechanism by which main root resistance operates, although it does not materially contribute to the resistance of Vertou and Sytan. Antibiosis has previously been assigned to carrot on the basis of differential invasion of roots from inoculation with equal numbers of eggs (Guerin et al., 1981). However, the result did not preclude differential invasion due to nonpreference behavior by the larvae in response to root chemicals. The present data provide an unambiguous demonstration of antibiosis. As this mechanism takes effect through physiological inhibitors, toxins, and decreased nutrient levels (Beck, 1965), it is reasonable to implicate root chemicals in its action against the Psila larva. In summary, the present data identify root-mediated resistance as operating by: (1) eliciting fewer eggs (nonpreference by the female), (2) independently decreasing larval invasion (nonpreference by the larvae), and (3) antibiosis against the root-established larvae. Accordingly, root-mediated resistance has a pervasive role in carrot resistance to Psila. Root chemicals are already established as contributing to the second of these mechanisms (Guerin and Ryan, 1984), but it is now apparent that they also contribute to both the former and the latter. Accordingly, main root chemical constituents may be crucial in carrot resistance to Psila. Acknowledgments--We thank Dr. P.R. Ellis, National Vegetable Research Station, Wellesbourne, U.K., for supplying seed and some statistical analyses.
REFERENCES ~xHLBERG, O. 1944. Olika morotsorter och morotflugan. Vaextskyddsnotiser. 8:49-50. BECK, S.D. 1965. Resistance of plants to insects. Annu. Rev. Entomol. 10:207-232. BEIRNE, B.P. 1971. Pest insects of annual crop plants in Canada: Psila rosae (F.), the carrot rust fly. Mere. Entomol. Soc. Can. 78:63-65. CAMeBELL, C.A.M. 1983. Antibiosis in hop (Humulus lupulus) to the damson-hop aphid, Phorodon humuli. EntomoL Exp. Appl. 33:57-62. DUNCAN, D.B. 1955. Multiple range and multiple F test. Biometrics 11:1-42. ELLIS, P.R., and HARDMAN, J.A. 1981. The consistency of the resistance of eight carrot cultivars to carrot fly attach at several centres in Europe. Ann. Appl. Biol. 98:491-497. ELLIS, P.R., WHEATLEY, G.A., and HARDMAN,J.A. 1978. Preliminary studies of carrot susceptibility to carrot fly attack. Ann. Appl. Biol. 88:159-170. FREULER, J., and FISCHER, S. 1983. Le Piege a Oeufs, nouveaux moyen de prevision d'attague pour la mouche du chou, Delia radicum (brassicae) L. Rev. Suisse Vitie. Arboric. Hortic. 15:107-110.
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GORHAM, R.P. 1934. Control of carrot mst fly, Psila rosae Fab. Rep. Que. Soc. Prot. Plants 26:90-96. GUERIN, P.M., and RVAN, M.F. 1980. Insecticidal effect of trans-2-nonenal, a constituent of carrot root. Experientia 36:1387-1388. GUERIN, P.M., and RYAN, M.F. 1984. Relationship between root volatiles of some carrot cultivars and their resistance to the carrot fly, Psila rosae. Entomol. Exp. Appl. 36:217-224. GUERIN, P.M., and ST.~DLER, E. 1982. Host odour perception in three phytophagous Diptera--a comparative study, pp. 95-105, in J.H. Visser and A.K. Minks (eds.). Proceedings of the 5th International Symposium on Insect-Plant Relationships. Pudoc, Wageningen. GUERIN,P.M., GFELLER,F., and ST,~DLER,E. 1981. Carrot resistance to the carrot fly--Contributing Factors. Report o f the Second Meeting, Eucarpia/IOBC Working Group Breeding for Resistance to Insects and Mites. West Pal. Reg. Sect. Bull. IV:63-65. JONES, O.T. 1979. The responses of carrot fly larvae, Psila rosae, to compounds of their physical environment. Ecol. Entomol. 4:327-334. OVERBECK,H. 1978. Untersuchnngen zum Eiblage- und Befallsuerholten der Mohrenfiiege, Psila rosae F. (Diptera: Psilidae), im Hinblick auf eine modifizierte chemische Bekampfung. Mitt. Biol. Bundesanstalt Berlin-Dahlem Heft. 183:1-183. RYAN, M.F., and GUERIN, P.M. 1982. Behavioural responses of the carrot fly larva, Psila rosae, to carrot root volatiles. Physiol. Entomol. 7:315-324. RYAN, M.F., GUERIN, P.M., and BEHAN, M. 1978. Possible roles for naturally occurring chemicals in the biological control of the carrot fly, pp. 130-153, in J.J. Duggan (ed.). Biological Control. Symposium of the Royal Irish Academy. SOUCHEY,J.F. 1970. Laboratory methods for work with plant and soil nematodes. Technical Bulletin (No. 2). M.A.F.F., HMSO, London. STADLER, E. 1971. Uber die Orientierung und das Wirtswahlverhalten der Mohrenfliege, Psila rosae F. (Diptera:Psilidae) I. Larven. Z. Angew. Entomol. 69:425-438. VAN'T, SANT, L.E. 1961. Levenswijze en bestrijding van de wortelvlieg (Psila rosae F.) in Nededand. Versl. Landbouwk. Onderz. 67:1-131. WHITCOMB,W.D. 1938. The carrot rest fly. Bull. Mass. Agric. Exp. Stn. 352:1-36. WRIGHT, D.W., and ASHBY, D.G. 1946. Bionomics of carrot fly. I. The infestation and sampling of carrot crops. Ann. Appl. Biol. 33:69-77.
