Journal of Nematology 29(3):306-314. 1997. © The Society o f Nematologists 1997.
Effect of Temperature on Pratylenchus penetrans Development TAKAYUKIMIZUKUBO 1 AND HIROSHI ADACHI 2 Abstract: Reproduction and development o f Pratylenchuspenetrans were studied on genetically transformed ladino clover roots. Solitary females developing on transformed roots in nutrient gellan gum m e d i u m (pH 5.5) deposited 1.2, 1.5, 1.6, 1.8, and 2.0 eggs per day at the respective temperatures of 17, 20, 25, 27, and 30 °C. The n u m b e r of eggs deposited was highly correlated with temperature. A reduction in egg-laying rates at the start of hatching was observed at all temperatures.Juvenile mortality was higher at 17 °C (50.4%), 20 °C (50.3%), and 30 °C (58.4%) than at 25 °C (34.6%) and 27 °C (37.6%). Life-cycle (egg deposition to egg deposition) duration was 46, 38, 28, 26, and 22 days at the respective temperatures. The developmental zero degrees (°C) and the effective accumulative temperatures (degree-days) required for hatching, female emergence, and onset of oviposition (completion of one generation) o f P. penetrans were estimated to be 2.7 and 200, 4.2 and 548, and 5.1 and 564, respectively. Pratylenchuspenetrans reproduces over a wide range o f temperatures. Key words: degree-day, developmental zero degree, egg-laying rate, effective accumulative temperature, ladino clover, life cycle, nematode, Pratylenchuspenetrans, reproduction, transformed hairy root.
Pratylenchus penetrans (Cobb) Filipjev & Schuurmans Stekhoven is recorded on more than 350 hosts and is distributed widely throughout temperate areas of the world (Corbett, 1973). In Japan, this nematode mainly has been reported from areas with an annual mean atmospheric temperature of 15 °C or less (Gotoh, 1974). The rate of development of P. penetrans was nearly a linear function of temperature, and higher temperatures (30 °C and 33 °C) affected fecundity (Mamiya, 1971). Although this species completed its life cycle by 35 days at 21 °C on both potato and onion plants (Wong and Ferris, 1968), optimum temperature for reproduction varied depending on the test plants (Acosta and Malek, 1979; Dickerson et al., 1964; Griffin, 1993; Kimpinski and Willis, 1981). Soil pH (Kimpinski and Willis, 1981; Morgan and MacLean, 1968; Willis, 1972) and moisture (Kable and Mai, 1968) also affected reproduction of P. penetrans. Because soil temperature affects activities of nematodes and greatly influences or regulates other parameters (Norton, 1978), thermal studies on the functions of reproducReceived for publication 1 November 1996. i Laboratory of Plant Nematology, Kyushu National Agricultural Experiment Station, Suya 2421, Nishigoshi, Kumamoto, 861-11 Japan. 2 Biological Science Research Center, Lion Co., Tajima 100, Odawara, Kanagawa, 256Japan. E-mail:
[email protected] p The authors thank N. Ishibashi (Saga University) and J.T. Gaspard (Nematec Co.) for reviewing the manuscript.
306
tion, female maturity rate, egg-laying rate, mortality of each stage, and duration of oviposition have primary importance. High temperatures apparently accelerated P. penetrans female development (Mamiya, 1971). The egg-laying rate of P. penetrans has been studied at 23 °C (Zunke, 1990b) and at various other temperatures (Mamiya, 1971). However, the effect of temperature on the duration of egg-laying and mortality is unknown. In addition, the developmental zero degree and effective accumulated temperature r e q u i r e d for P. penetrans to pass through each developmental stage have not been reported. The objective of this study was to identify how parameters such as egg-laying rate, hatching rate, mortality ofjuveniles, and duration of life cycle of P. penetrans vary with temperature. MATERIALS AND METHODS
Experiments were conducted using genetically transformed Triforum repens L. race giganteum (Ladino clover) roots, as application of transformed roots to the study of the life cycle in Meloidogyne species has proven successful (Adachi et al., 1992, 1993). Transformed roots produced according to Adachi et al. (1993) were maintained on Woody Plant Medium (WP) (Lloyd and McCown, 1980). Fifteen-ram-long root tips were placed in 4 ml of root culture media (RM)
Pratylenchus penetrans Development: Mizukubo, Adachi 307 (Becard a n d Fortin, 1988) in 35- x 10-mm dishes were sampled f r o m 17 a n d 25 °C, 20 a n d 27 °C, a n d 30 °C, respectively, at each tissue culture dishes (Falcon 3001, Becton interval after inoculation. R e m o v e d roots Dickinson, produced by Radie Inc., Takasaki, G u n m a , J a p a n ) . Each r o o t was were s t o r e d in t r i e t h a n o l a m i n e - f o r m a l i n (TAF) preservative until they were studied. incubated at 25 °C for at least 24 hours until inoculation. T h e Pratylenchus penetrans cul- N e m a t o d e s or eggs p r e s e n t in the substrate ture used (Chiba-1 isolate) originally was were c o u n t e d after roots were r e m o v e d . collected f r o m a f a r m e r ' s c a r r o t field in Each root preserved in TAF was washed with F u n a b a s h i City, Chiba, J a p a n , a n d main- tap water three times, a n d placed in 3-ml tained on carrot in the g r e e n h o u s e . A p u r e glass tubes with 1 ml tap water. Roots a n d n e m a t o d e s were stained with an NaOCl-acid isolate f r o m a k i d n e y b e a n p o t culture, which h a d b e e n p r o p a g a t e d f r o m a single fuchsin-glycerin m e t h o d (Byrd et al., 1983). female a n d seven males obtained f r o m the Roots were t h e n pressed between two pieces of glass and observed. carrot p o t culture, was aseptically inoculated An e x p e r i m e n t a l plan developed by G a d d to l u c e r n e callus i n d u c e d by m o d i f i e d Schenk a n d H i l d e b r a n d m e d i u m (Mitsui, a n d Loos (1941) a n d Mamiya (1971) was 1977), t h e n cultured for a year as a m o n o x - used to estimate the onset o f events in the enic callus culture. I n o c u l u m was extracted life cycle o f P. penetrans inside roots. Instead for 24 hours with B a e r m a n n funnels (Mitsui, o f an overall m e a n , estimation of fecundity 1974). N e m a t o d e s were washed 3 times with was taken f r o m the m e a n s of data sets that sterile tap water. A female was micropipet- excluded the lower half of the values and ted o n t o RM in a few microliters of water the m a x i m u m value f r o m the entire data a b o u t 10 m m f r o m the root. After inocula- sets. All regression analyses, except early fertion, culture dishes were sealed with a strip tility (eggs laid), w e r e p e r f o r m e d u s i n g o f Parafilm " M " (American National Can, these u p p e r - h a l f m e a n s o f the data sets. ReGreenwich, CT). Culture dishes were kept in gression lines for early fertility (eggs laid) an incubator at 25 °C for 24 hours for the were calculated using m e a n s of entire replie x p e r i m e n t s at 17 to 27 °C. N e m a t o d e s in- cations. T h e onset (day) o f egg deposition side culture dishes were t h e n observed with a n d juvenile hatch by the first female were a dissecting m i c r o s c o p e , a n d dishes with d e t e r m i n e d by interpolation o f the regresfree females or d e a d females outside r o o t sion equations to the abscissa (day). T h e tips were discarded. T h e r e m a i n d e r were first fertility (eggs laid) of the n e x t generakept in incubators at 17 + 1, 20 + 1, 25 + 0.5, tion was a m e a n of groups o f eggs that were a n d 27 + 1 °C. These e x p e r i m e n t s started 24 o b s e r v e d in r e m o t e loci f r o m the initial hours after the real inoculation. Unlike ex- colony. T h e n , the onset (day) of egg depop e r i m e n t s at o t h e r temperatures, the experi- sition by the n e x t g e n e r a t i o n was m e a s u r e d m e n t at 30 °C was started soon after inocu- by squaring a line having the regression colation, a n d kept in an incubator at 30 + 1 °C. efficient of the early fertility (eggs laid) in Dishes with free females or d e a d females the first generation a n d passing t h r o u g h the outside r o o t tips were discarded after 24 n e x t g e n e r a t i o n ' s fertility at the first obhours. T o reduce possible detrimental ef- served day. fects o n n e m a t o d e r e p r o d u c t i o n d u e to shortage o f n o u r i s h m e n t , roots kept at 20, RESULTS 25, 27, a n d 30 °C were r e m o v e d f r o m tissue For all t e m p e r a t u r e s , 88% o f initial feculture dishes after 14 days; those k e p t at 17 males were present in r o o t tissue at 2, 4, 7, °C were r e m o v e d 21 days after inoculation 10, a n d 14 days after inoculation, but often a n d transferred to dishes filled with fresh m e d i u m . E x p e r i m e n t s at 17, 20, 25, a n d 27 d i s a p p e a r e d at 17 days a n d thereafter. Fertility of females u n d e r identical t e m p e r a t u r e °C were c o m p o s e d o f two o b s e r v a t i o n s , while a single e x p e r i m e n t was c o n d u c t e d at a n d sampling periods was less variable until 30 °C. Twelve to 16, 8 to 16, or 8 to 12 petri juveniles started to hatch, b u t was increas-
308 Journal of Nematology, Volume 29, No. 3, September 1997 ingly variable thereafter. In general, small, stained particles were observed near eggs and juveniles when there were fewer nematodes inside roots. A few females stayed in the culture substrates or were found in the TAF that was used as root preservative. During the first 2 weeks after inoculation, females were observed in roots at their basal trunk, and clusters of eggs a n d / o r juveniles were observed near the females. At the end of the experiment, besides numerous eggs in the first established cluster, more than one group of eggs with or without a female were observed in remote sites. Before maturing, some nematodes migrated from the original sites to colonize remote sites of the root. Tracks of nematode movement on the substrate were observed at all temperatures after 35 days (17 °C), 32 days (20 °C), 14 days (25 °C), 10 days (27 °C), and 17 days (30 °C). The migration of nematodes was frequent on transformed ladino clover roots b u t rare in preliminary tests with transformed lettuce roots. This characteristic of transformed clover roots proved useful for the study of the life cycle, as the eggs deposited separately by the next generation of females permitted estimation of the first oviposition date by the second-generation females. Oviposition occurred 4 days after inoculation at 17 °C (Fig. 1A), but was 2 days after inoculation at the other four temperatures (Fig. 1B-E). There were steady increases in egg numbers for the first 17, 14, 10, 7, and 7 days at 17, 20, 25, 27, and 30 °C, respectively (Table 1, Fig. 2). The oviposition rates during this period were 1.2, 1.5, 1.6, 1.8, and 2.0 eggs per day at the respective temperatures of 17, 20, 25, 27, and 30 °C (Table 1, Fig. 2). There was a significant correlation between the egg-laying rate of P. penetrans and temperature (Fig. 2). The regression lines at 17, 20, 25, 27, and 30 °C had an estimated onset of oviposition of 2.7, 1.6, 1.1, 0.7, and 1.6 days after inoculation, respectively. The rate of oviposition (eggs per day) at the experimental temperatures diminished to 0.3 (17 °C), 0.3 (20 °C), 0.4 (25 °C), 1.6 (27 °C), and 0.8 (30 °C) at 17, 14, 10, 7, and 7 days after inoculation, respectively.
Juveniles were first seen 17 days after inoculation at 17 and 20 °C (Fig. 1A,B), 14 days at 25 °C (Fig. 1C), 10 days at 27 °C (Fig. 1D), and 14 days at 30 °C (Fig. 1E), then steadily increased thereafter. The estimated onsets of juvenile hatch determinated from the regression equations were 17.5, 13.1, 9.7, 8.8, and 9.2 days after inoculation at the experimental temperatures of 17 to 30 °C, which coincided with the days when rates of oviposition diminished at the five temperarares (Fig. 1A-E). The estimated embryonic periods decreased as temperature increased: 14.7 days (17 °C), 11.4 days (20 °C), 8.6 days (25 °C), 8.1 days (27 °C), and 7.6 days (30 °C) (Table 1). Adults at 17 °C were n o t observed until day 49, and the best approximation to adult emergence was 45.5 days after inoculation (Fig. 1A). At 20 °C, males first appeared on day 35, but females were not seen until day 39 (Fig. 1B). Thus, appearance of females at this temperature must have been between days 35 and 39. At 25 °C, males and females first appeared on days 25 and 28, respectively, and female appearance was between days 25 and 28 (Fig. 1C). At 27 °C, males first appeared on day 25, and a possible next-generation female also was first observed on day 25 (Fig. 1D). Adults at 30 °C appeared on day 25, and the appearance of females was between days 21 and 25 (Fig. 1E). Total juvenile development times for females were approximately 28.0 days (17 °C), 23.9 days (20 °C), 16.7 days (25 °C), 16.2 days (27 °C), and 13.8 days (30 °C). The duration from eggs to adult females were 42.7, 35.4, 25.4, 24.3, and 21.4 days for the respective temperatures of 17, 20, 25, 27, and 30 °C (Table 1). At respective temperatures of 17, 20, 25, 27, and 30 °C, maxima of 30, 34, 30, 46, and 31 offspring (including eggs) were laid by one initial female in 35, 35, 17, 17, and 21 days, although maxima of 107, 69, 115, 127, and 40 offspring were produced during the incubation period (Table 1). The next generation of females laid 4.7 eggs on day 53 (17 °C), 2.9 eggs on day 42 (20 °C), 4.6 eggs on day 32 (25 °C), 1.8 eggs on day 28 (27 °C), and 2.0 eggs on day 25 (30 °C).
Pratylenchus penetrans Development: Mizukubo, Adachi 309
90
80
A
80
= --•
y= -3.37+1.23x r 2= 0.993 ***
70
- -y= 13.29+0.33x r2= 0.953 ***
70
-- -& -- y= -10.6+0.61x r 2= 0.991 ***
60
-- -e -- y= 23.85-0.28x r 2= 0.956 *** JI,
60 50
y= -60.49+1.23x
20
20
10
10
0
v
0
-- -& -- y= -9.93+0.76x
r z= 0.957 ***
-- -O-- y= 24.08-0.41x
r 2= 0.835 **T
4,
y= -61.38+1.53x
90
L
/
80
100 / 90 I-
- ~-- ~ -91.60+4.26xr' =0.991** / - .A - y=.10.26+1.o6xr==0.970** /
70
-- " e - - y= 21.32-0.69X r 2 = 0 . 8 4 5 *
60
l-
J.
- - o --y=-74.31+3.31x?= 0.977(/T
50 40
f
~..
~ ~
D
14 21
28
35 42 49
-1.31+1.78x r2= 0.993 * .--- -y="p -1.23+1.57x r2= 0.961 *
--•
-A
-- -& -- y= -9.82+1.11x r 2= 0.997 * -- -e -- y= 3.58+0.79x r 2= 0.997 NS .e y= -48.04+1.78x
IE ~;i;o o A
' 7
E=
40
7
40 30
/
50
"'~"
5O
.
0
=-8-
0
7 14 21 28 35 42 49 56 63 = y=-1.81+1.62x?:o.993* T
70 I"
-
0
u
120 110 [ C
0
r 2= 0.997 ***
30
30
,~
y=-2.49+1.53x
- -y= 14.78+0.32x r 2= 0.887 ***
40
40
80
• --•
50
~.~
B
14
21
28
35
0
42
0
•
y=-3,16+1.97X
r 2= 0.987 NST
- - O - -y = 4.13+0.81x
r2= 0.872 * 1
-- -& -- y= -7,57+0.82x
r 2= 0,983 * Ir
- - o
-- - 0 - - y= 10.34+0,07x
r z= 0,016 NS I
-
¢ 4-
~"~ 7 14
21
28
35
42
Total fecundity. First linear i n c r e a s e . - - S e c o n d linear increase. - Third linear increase.
o
Eggs.
z~
Hatched juveniles.
•
E g g s laid b y n e x t g e n e r a t i o n f e m a l e s .
[:]
First r e c o v e r y d a y o f m a l e s a l o n e .
•
First r e c o v e r y d a y o f n e w f e m a l e a n d males.
0 0
7
14
21
28
Days after inoculation FIG. 1, T i m e course c h a n g e s in total fecundity, h a t c h e d juveniles, a n d eggs of a single Pratylenchus penetrans female o n t r a n s f o r m e d ladino clover roots. A) 17 °C. B) 20 °C. C) 25 °C. D) 27 °C. E) 30 °C. Bars show standard deviation for total fecundity. NS: n o t significant.
310 Journal of Nematology, Volume 29, No. 3, September 1997 TABLE 1.
Comparative population dynamics and development of
Pratyl~nchzts penet,rans o n
transformed root
explants and whole-seedling experimental systems at various temperatures. Transtbrmed ladino clover root system~ (Present study) 17 °C
Egg-laying rate per dayb Maximal numbers of offspring during the incubation period (days) Embryonic development (days) Postembryonic development (days) Duration from egg to adult Duration from egg to egg
20 °C
1.23 107 (63)
1.53 69 (46)
25 °C
27 °C
1.62 115 (39)
1.78 127 (42)
Conifer seedling system (Mamiya, 1971)
30 °C
1.97 40 (25)
15 °C
0.71
20 °C
1.46
24 °C
1.25
30 °C
1.21
33 °C
0.09
49 (_)c
58 (35)
53 (42)
39 (28)
---
14.7
11.4
8.6
8.1
7.6
25
14-16
8-10
9-10
--
28.0
23.9
16.7
16.2
13.8
53
24
16
17
--
42.7
35.4
25.4
24.3
21.4
78
38
26
27
--
46.4
38.5
28.1
26.2
22.4
86
35
30-31
--
42-44
a Each temperature with 8 to 16 replications. b Rate d e t e r m i n e d d u r i n g a steady egg-laying period 7 to 17 days after inoculation. e Duration in days not given.
T h e life cycle o r g e n e r a t i o n t i m e o f P. penetrans w e r e e s t i m a t e d to b e 46.4, 38.5, 28.1, 26.2, a n d 22.4 days a t 17, 20, 25, 27, a n d 30 °C, respectively. G e n e r a t i o n t i m e o f P. penetrans (28.1 days at 25 °C) m a y b e a n u n d e r e s t i m a t e b e c a u s e t h e n u m b e r s o f eggs alr e a d y h a d i n c r e a s e d r a p i d l y 28 days a f t e r ino c u l a t i o n (Fig. 1C).
2.2 "0
2.0 I
y = 0.4068+0.0512f r 2 = 0.944 (P < 0.01)
e~
E 1.8 W ~t N,=
0
6
Z
1.6 1.4 z~
1.2 1.0
I
15
I
I
20 25 30 T e m p e r a t u r e (°C)
FIG. 2. Changes of number of eggs deposited per female Pratylenchuspenetransper day as influenced by temperature. Nematodes were inoculated onto transformed ladino clover roots cultured in nutrient medium.
N e m a t o d e s o u t s i d e r o o t s at 17 °C w e r e first f o u n d o n d a y 35 (16% o f t o t a l n e m a t o d e s e x c l u d i n g eggs), l e v e l e d off at 6 - 1 0 % d u r i n g days 35 to 56, t h e n q u i c k l y i n c r e a s e d to 95% o n d a y 63 (Fig. 3). M a l e s a n d f e m a l e s w e r e f o u n d o u t s i d e r o o t s o n d a y 49 a n d t h e r e a f t e r . A t 20 °C, n e m a t o d e s w e r e n o t f o u n d o u t s i d e r o o t s u n t i l 28 days ( o n e m a l e o b s e r v e d ) , f l u c t u a t e d b e t w e e n 10 to 3 1 % o f t h e e n t i r e p o p u l a t i o n f r o m d a y 32 to 42, a n d i n c r e a s e d t h e r e a f t e r to a m a x i m u m o f 67% o n d a y 46 (Fig. 3). N e m a t o d e s at 25 °C w e r e n o t s e e n o u t s i d e r o o t s u n t i l 14 days, w h e n 29% o f all j u v e n i l e s w e r e o b s e r v e d . N e m a t o d e s w e r e n o t s e e n o u t s i d e r o o t s o n days 17 a n d 21 a f t e r i n o c u l a t i o n ; however, t h e y inc r e a s e d o n d a y 25 a n d t h e r e a f t e r , r e a c h i n g a l m o s t 100% o n d a y 35 (Fig. 3). A t 27 °C, m a n y j u v e n i l e s (33%) w e r e f o u n d o u t s i d e r o o t s o n d a y 14. T h e p e r c e n t a g e o f n e m a todes outside roots fluctuated between 4% a n d 23% f r o m d a y t 7 to d a y 39, a n d t h e n i n c r e a s e d to 68% o n d a y 42 (Fig. 3). A t 30 °C, n e m a t o d e s o u t s i d e r o o t s w e r e n o t s e e n u n t i l d a y 21, w h e n 13% o f all n e m a t o d e s were observed on the medium surface. N e m a t o d e s o u t s i d e r o o t s t h e n i n c r e a s e d to 26% o n d a y 25 a n d to 36% o n d a y 28. Eggs at all t e m p e r a t u r e s u s u a l l y w e r e l a i d
Pra~ylenchus penetrans Development: Mizukubo, Adachi 311 T h e r e was a significant correlation between the rates o f d e v e l o p m e n t (y) and temperature (x) (Table 2). T h e regression equation was y = -0.0090 + 0.001772x. T h e temperature of 5.08 °C was the point where the line intersected the abscissa and thus was the estimated developmental zero point o f P. penetrans for completion o f its life cycle. The total effective temperature (K) required to complete a given developmental stage was expressed as follows:
o 100
8
o
"O 100 [ j "~ ... 50 25 °C O
0
O
50 0
. . . . .
K=(V-A)× *--
-
-
E 100 [ _ ~ ~ : ~ , Z 50 30 °C 0
I
!
I
I
I
T;
where V= incubation temperature, A = developmental zero degree, T = days required to pass t h r o u g h a stage.
Pratylenchus penetrans required 564 degreedays to complete its life cycle (egg to egg). T h e developmental zero point (°C) and the FIG. 3. Time course changes of hatched Pratylenchus total effective temperature (degree-days) repenetrans outside roots as affected by temperature. The quired to pass through the respective devely-axis denotes population (juveniles a n d adults) outside opmental stages, including c o m p l e t i o n of roots as percentage of total population. Nematodes are the life cycle, are p r e s e n t e d in Table 2. T h e the descendants of a single female in a transformed ladino clover root cultured in nutrient medium. d e v e l o p m e n t a l zero d e g r e e s differed dep e n d i n g o n stages: 2.74 for passing through inside roots. Less than 3% o f all eggs at 17 e m b r y o n i c period; 4.18 for adult female °C were f o u n d outside roots at 21, 35, and emergence. Hatching and female matura63 days after inoculation. A few eggs (2%, tion required 200 and 548 degree-days, re3%, and 1% of all eggs, respectively) were spectively. also f o u n d on day 46 at 20 °C, day 35 at 25 DISCUSSION °C, and day 25 at 30 °C, respectively. However, at 27 °C, although only 2% and 1% o f We observed that variability of n e m a t o d e eggs were outside roots o n day 17 and 28, as populations at all temperatures became inmany as 12% o f eggs were outside roots on creasingly large during the experiments. Because all the nematodes outside roots were day 42. Rate o f d e v e l o p m e n t p e r day for each counted, the lower fertility in some replicatemperature was given as a reciprocal of the tions might be attributed to lower n e m a t o d e days r e q u i r e d to c o m p l e t e the life cycle. populations inside roots due to death o f
0 7 14 21 28 35 42 49 56 63 Days after inoculation
TABLE 2. Developmental zero degrees (°C) and total effective degree-days required to pass t h r o u g h three different developmental stages of Pra~lenchuspenetrans. Equation ~ Incubation period (Egg-deposition to hatch) Female emergence period (Egg deposition to adult female) Life-cycle duration (Egg deposition to egg deposition)
y = -0.0137 r2 = 0.9773 y = -0.0076 r~ = 0.9928 y = -0.0090 r~ = 0.9966
a y = velocity of development, x = temperatures (°C).
+ 0.005004x (P --< 0.01) + 0.001824x (P--< 0.001) + 0.001772x (P -- 0.001)
Developmental zero (°C)
Degree-days
2.74
200
4.18
548
5.08
564
312 Journal of Nematology, Volume 29, No. 3, September 1997 nematodes. Nusbaum and Barker (1971) considered that the mortality rate in phytoparasitic n e m a t o d e populations might be related m o r e to physiological responses of the host than to n e m a t o d e density. In this study, we observed little necrosis (hypersensitive response) in ladino clover roots. However, Sawhney and Webster (1979) suggested the presence of o t h e r mechanisms of resistance besides the h y p e r s e n s i t i v e r e s p o n s e o n nematode-resistant tomato. Similarly, Veech (1981) noticed the presence of hypersensitivity without browning. Fate of the dead nematodes inside roots has never been illustrated, but we could observe the collapsed o r s h r u n k e n eggs a n d j u v e n i l e s o f Pratylenchus coffeae inside sterile t r a n s f o r m e d soybean roots (unpubl.). Thus, it seems reasonable to hypothesize that plant roots have the ability to d e c o m p o s e dead nematodes. This hypothesis helps to explain why low n e m a t o d e populations inside ladino clover roots often were accompanied by n u m e r o u s stained particles. Given a d e c o m p o s i t i o n ability of host roots, it seems reasonable to interpret the increasing variability of nematodes as correlated with increasing fluctuation o f a root defense response. Thus, in the present regression analysis, we chose to use the means of upper-haK data of the replications, expecting them to be adequately representative of potential fecundity. The present study confirms the limited mobility of P. penetrans females inside roots (Mamiya, 1971). Females stayed n e a r the sites where they entered after inoculation for at least 2 weeks. T h e presence o f nematodes outside roots (Fig. 3) suggests that the m o v e m e n t o f nematodes from the initial infestation took place through the medium, which presumably would be through soil under natural conditions. Zunke (1990b) observed that when P. penetrans p e n e t r a t e d roots, it subsequently exited the same hole and moved to a n o t h e r area o f the root. Observation of a 1- to 2-week lag from juvenile hatching to the first peak of nematodes outside the root at lower and h i g h e r temperatures (17, 20, and 30 °C) is similar to results from P. scribneri (MacGuidwin, 1989), that the older stages (14, adult) are more likely to
leave roots than J2 and J3. Conversely, the first peak o f juveniles outside roots at 25 and 27 °C o c c u r r e d i m m e d i a t e l y after t h e i r hatching, indicating that migration activity of second-stage P. penetrans was greatest at 23-27 °C. However, an extremely high seco n d peak of P. penetrans outside roots at the end of every e x p e r i m e n t can be attributed to the nematode's response to root aging in the exhausted medium. MacGuidwin (1989) estimated 20-50% o f field plot populations of P. scribneri to be in the soil. T h e present observation o f eggs outside roots, along with ectoparasitic feeding behavior (Kurppa and Vrain, 1985; Zunke, 1990a), suggests that, as in Pratylenchus ag~lis (Rebois and Huettel, 1986), some P. penetrans can complete their life cycle outside roots as ectoparasites. T h e rate of development of P. penetrans was f o u n d to be a linear function o f temperature. This relationship also exists in Ditylenchus dipsaci (Griffith et al., 1996) and in species of the genera Meloidogyne, Heterodera, Globodera, Longidorus, Goodeyus, a n d Caenorhabditis (Trudgill, 1995). Egg-laying rate and development of P. penetrans from 17 to 30 °C were c o m p a r e d with results from a conifer (Cryptomeria japonica) seedling system (Mamiya, 1971) (Table 1). Mamiya (1971) f o u n d that higher temperatures (30 °C and 33 °C) suppressed the fecundity o f P. penetrans; in the present study, 30 °C did not suppress fecundity. T e m p e r a t u r e and number of eggs deposited (re = 0.944; P--< 0.01) were highly correlated, with a maximum of 2.0 eggs deposited per day at 30 °C (Fig. 2, Table 1). High fecundity of P. penetrans at 30 °C observed in this study helps to explain why this n e m a t o d e often has been f o u n d in southern Japan (unpubl.) and in subtropical and tropical regions such as India and the Philippines (Corbett, 1973), Algeria ( L a m b e r t i et al., 1975), Egypt (Oteifa, 1962), and Brazil (Swarup and Sosa-Moss, 1990). O t h e r thermotypes of this species may occur (Gotoh, 1974). A s u d d e n decline in egg-laying rate at each temperature was a c o m m o n characteristic o f this and a previous study (Mamiya, 1971). This p h e n o m e n o n c a n n o t be attributed to a reduction in the egg-laying rate
Pratylenchus penetrans Development: Mizukubo, Adachi 313 after production of a given n u m b e r of eggs because the accumulated numbers of eggs (egg-laying rate x days) at these turning points did not result in constant values: 21, 21, 16, 18, and 14 eggs at 17, 20, 25, 27, and 30 °C, respectively. We interpret the phen o m e n o n as the loss of juveniles after hatching because the turning point coincides with the onset of juvenile hatching at each temperature, and a juvenile regression line had lower regression coefficients than an early egg-laying line (Fig. 1A-E). Thus, the juvenile population was lost at a certain rate after hatching. Assuming the lost juveniles inside the roots decomposed after their death, as already described, the early juvenile mortality (death rate) can be computed by the following formula: Mj = 1 - (g/Re) ;
where, Mj = mortality of juvenile, Rj = regression coefficient in the juvenile regression equation, R e = regression coefficient in the early fertility (eggs laid) regression equation. Mortality of the juveniles thus computed was 50.4, 50.3, 34.6, 37.6, and 58.4% at 17, 20, 25, 27, and 30 °C, respectively. We assumed that this mortality is due to temperature effects because host response effects were removed when we chose to use the mean of the upper half of the replications (excluding the m a x i m u m value) as the potential fertility. Juvenile mortality was lower (one-third of fertility) at 25 and 27 °C than at 17, 20, and 30 °C (half of fertility), providing a possible explanation for why temperatures of about 25 °C are optimal for the m u l t i p l i c a t i o n of P. penetrans on c o r n ( D i c k e r s o n et al., 1964) a n d s o y b e a n (Acosta a n d Malek, 1979). Conversely, Dunn (1973), Acosta and Malek (1979), and Griffin (1993) reported highest reproduction on alfalfa at 30 °C, and Dickerson et al. (1964) reported 16 °C to be the optimum on potato. Reproduction of Pratylenchus appears to d e p e n d on the nematode-host int e r a c t i o n , n o t o n l y on t h e n e m a t o d e (Dickerson, 1979). Temperature also affects
plant resistance to nematodes, and resistance is often ineffective at high temperatures (Brueske a n d Dropkin, 1973; Okamoto and Mitsui, 1977). Our results confirm the importance of temperature to the development of P. penetrans. Temperature affects egg-laying rate and juvenile mortality, probably through interaction with other factors. The information also is valuable in forecasting the occurrence of P. penetrans. The effects of temperature on longevity or the egg-laying period of female P. penetrans remain to be investigated. LITERATURE CITED Acosta, N., and R. B. Malek. 1979. Influence of temperature on population development of eight species of Pratylenchus on soybean.Journal of Nematology 11:229232. Adachi, H., T. Narabu, andY. Momota. 1992. Culture of Meloidogyne incognita on oriental-melon roots genetically transformed by Agrobacterium rhizogenes. Japanese Journal of Applied Entomology and Zoology 36:225230. Adachi, H., T. Narabu, andY. Momota. 1993. Culture of Meloidogyne hapla and M. javanica on several plant roots genetically transformed by Agrobacterium rhizogenes. Japanese Journal of Nematology 23:63-70. Becard, G., and J.A. Fortin. 1988. Early events of vesicular-arbuscular mycorrhiza formation on Ri TDNA transformed roots. New Phytologist 108:211-218. Brueske, C. H., and V. H. Dropkin. 1973. Free phenols and root necrosis in Nematex tomato infected with the root-knot nematode. Phytopathology 63:329-334. Byrd, D.W., Jr., T. Kirkpatrick, and K.R. Barker. 1983. An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology 15:142-143. Corbett, D. C. M. 1973. Pratylenchuspenetrans. C. I. H. descriptions of plant-parasitic nematodes. Set 2, No. 25. Walling-ford, UK: Commonwealth Agricultural Bureaux. Dickerson, O.J. 1979. The effect of temperature on Pratylenchus scribneri and P. alleni populations on soybean and tomato. Journal of Nematology 11:23-26. Diekerson, O.J., H.M. Darling, and G.D. Griffin. 1964. Pathogenicity and population trends of Pra~lenchus penetrans on potato and corn. Phytopathology 54:317-322. Dunn, R.A. 1973. Effect of temperature on survival and reproduction of Pratylenchuspenetrans (Cobb, 1917) Filipjev and Schuurmans-Stekhoven, 1941. Ph.D. dissertation, Cornell University, Ithaca, New York. Gadd, C. H., and C. A. Lots. 1941. Observations on the life history of Anguillulina pratensis. Annals of Applied Biology 28:39-51. Gotoh, A. 1974. Geographic distribution of Pratylenchus spp. (Nematoda: Tylenchida) in Japan. Bulle-
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