A. Ben-Aziz el .I. (19741 Pfiytop.rasitir

2(2): 117-125,

PERSISTENCE AND DISTRIBUTION OF THREE BENZIMIDAZOLE FUNGICIDES IN PEAR TREES SPRAYED FOR THE C O N T R O L OF

VENTURIA PIRINA

By A.

B E N - A Z l z ' : . E . SHAB[' AND

N.

AHARONSON-"

The persistence and distribution in leaves and fruits of benomyl, carbendazim and meihylthiophanate were investigated, following fungicide spray treatments in pear orchards. Chemical analysis and bioassays showed that basal leaves sprayed more than once with benomyl, contained 50 ppm of methyl benzimidazole cltrbamate IMBC) 4 days after the last spray. Seven m o n t h s later, 8 ppm MBC was detected in the basal leaves, jt,st before normal abscission. In apical leaves, the fungicide levels were lower than in the basal ones. Analyses of basal leaves showed that the residue level of MBC, 5 and 7 m o n t h s after the last treatment with benomyl, carbendazim or methylthiophanate, wits very similar in all treatments. Bound MBC was not detected in the leaves and only low concentrations of 2 - a m i n o benzimidazole (2-AB) were detected. There was no trans[ocation of these fungicides from treated leaves to new young leaves. Chemical analysis of pear fruits from an orchard sprayed with benomyl revealed that 3 weeks after the last spray treatment, the residue level of MBC was 0.4 ppm. About g 5 ~ of the fungicide was found in the peel, and only 15% in the pulp, The more infected fruits consistently contained a lower concentration of MBC than the less infected fruits. K E Y WORDS: Bcnzimidazole fungicides: residues: persistence: distribution: pear leaves and fruits: benomyl: MBC: methyl lhiophanate.

INTRODUCTION Pear scab caused by Venturia pirina is the most damaging disease to the Spadona variety of pear in Israel (1 I). Benzimidazole fungicides were found to be particularly effective in controlling the disease (6,9). Since dithiocarbamale fungicides are phytotoxic to the pear varieties grown in Publication of the Agricultural Research Organization, 1974 Series, No. 248-E. Received July 1974; accepted Sept. 1974. ~Killed in action on Oct. II, 1973, near the Suez Canal, Dr. Ben-Aziz had worked as Senior Researcher at the Pesticide C h e m i s t r y and Residue Research Laboratory, ARO, The Volcani Center, since its establishment in April 1971. 'Pesticide Chemistry and Residue Research Laboratory. and ~Div. of Plant Pathology, ARO, The Volcani Center, Bet Dagan.

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Israel and dodine increases fruit russeting in the Spadona variety, the benzimidazole derivatives are being used more and more for the control of pear scab. It has been shown that benzimidazole derivatives can prevent the development of the perithecial stage of Venturia spp. (3,4,5). The long-lasting protective effect against pathogenic fungi and the systemic activity of benzimidazole fungicides were reported widely (7,8). Trunk administration of MBC to pear trees was reported lately (10) but, in practice, the benzimidazole fungicides are applied in the orchard frequently as foliage sprays. Preliminary work (E. Shabi, unpublished data) showed that fungitoxic activity can be detected by bioassay in pear leaves collected from the tree or from the ground five months after spray treatment with one of several benzimidazole fungicides. In the present work we examined the persistence, accumulation, distribution and possible translocation of three benzimidazole derivatives applied as foliage sprays for pear scab control. MATERIALS AND METHODS The fungicides examined were: Methyl 1 - ( b u t y l c a r b a m o y l 2-benzimidazole carbamate (benomyl) (Benlate 50 W, E.I. du Pont de N e m o u r s and Company, Wilmington, Del., U.S.A.); 2-(methoxy carbamoyl)-benzimidazole (carbendazim) (Bavistin 50 W, BASF AG, Ludwigshafen, Germany); and 1,2-bis (3-methoxy-carbonyl-2-thioureido) benzene (methylthiophanate) (Topsin M 70W, Nippon Soda Co., Tokyo, Japan). Experiment with leaves Spray applications for pear scab control were made to 6-year-old Spadona pear trees. The fungicides were applied with an airblast sprayer, at the rate of 2,000 l/ha, which gave coverage to the point of spray run-off. During the spring, seven spray teatments were given on March 13 (green tip), 20, 28, April 6, 17, 27 and May 14. A treatment consisted of 30 trees (five rows of six trees each), replicated five times. Benomyl and carbendazim were applied at a concentration of 150 ppm a.i., and methylthiophanate at 210 ppm a.i. During the growing season, two series of leaf samples were taken from each treatment. In each series, leaves from extension shoots (terminals) were collected separately according to position (age) on the shoot: basal (oldest), apical (youngest), and intermediate. Each sample consisted of 18 leaves. One disc (13-mm diameter) was cut from each leaf and bioassayed for fungitoxic activity against Penicillium sp. by the agar plate method.

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Phytoparasitica 2:2, December 1974

In the series of samples collected during the spray program, the first sample was taken 9 days after the third spray, and the fourth and last sample was taken before the seventh and final application, which was also just before the end of the extension shoot growth. Additional samples were taken at two intermediate dates, both immediately before the spray application. A second series of samples was taken after the last spray application, to follow the rate of degradation of the fungicide in the leaves throughout the summer. By this time the basal leaves had received seven applications of the fungicides, and the apical leaves only one or two sprays. In this series, samples were collected on seven dates from 8 to 214 days after the last spray: May 22, June 1, 19, July 25, Oct. 24, Nov. 22, and Dec. 14 - - just before complete leaf fall. The second series of samples was subjected also to chemical analysis, which was based on fluorometric determination of MBC as described elsewhere (1). The sample underwent acetone extraction, partitioning between acetone-water and ethyl acetate, and clean-up on a magnesium oxide-celite-alumina column. The leaf samples weighed 20 g and were stored at -17~ until analysis. The chemical analyses were made on the following samples: benomyl-treated basal leaves collected on all seven sample dates; benomyl-treated apical leaves collected on the first five sample dates only; and carbendazim- and methyl thiophanate-treated basal leaves collected on the last three sample dates only (163, 192 and 214 days after the last spray). The analysis for 2-amino benzimidazole was carried out according to a method described elsewhere (2).

Experiment with fruit Pear fruits were collected from trees which had received a total of 13 sprays of benomyl at a concentration of 250 ppm a.i., applied with an airblast sprayer. Samples of 100 fruits were taken at random from a height of about 2 m at 2, 21, and 58 days after the last spray; the last sample of fruit was mature. Residues in healthy and diseased whole fruits were compared and the quantities were expressed as ppm MBC. In a harvest sample of healthy fruit, MBC residues were determined separately for peel and pulp. The fruit samples were stored and analyzed as described before. The result of the MBC residue in fruits is the average of three analyses, each consisting of a 300-g subsample. RESULTS

Bioassay of pear leaves during the early growing season Results for treated pear leaves collected in the spring during the growing season from different positions on the ektension shoots and

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l l9

bioassayed for fungitoxic activity, are presented in Table i. In the trees that were sprayed, fungitoxic activity was observed in the basal and intermediate leaves. Apical leaves that were I-3 days old and had emerged since the last spray did not show any activity. The fungitoxicity - expressed as diameter of the inhibition zone - - was very similar for all three fungicides. The activity in the basal leaves increased with the increase in the number of spray applications. TABLE 1 BIOASSAY OF PEAR L E A V E S SPRAYED WITH BENZIMIDAZOI~E F U N G I C I D E S

Sampling date Fungicide

No. o] l)ay~ .]ter Diameter of inhibition zone prerious h~t (ram) of Penicillium sp. sprays prccion~ applied in ~pray the orchard Apical* InterBasal leaves mediate leaves leaves

benomyl carbendazim methylthiophanate

April 6

3

9

0 0 0

31 23

35 36 24

benomyl carbendazim rnethylthiophanate

April 17

4

II

0 0 -

27 26

35 38 _

benomyl carbendazim methylthiophanate

April 27

5

10

0 0 0

36 3~; 38

4I 42 41

benomyl carbendazim methylthiophanate

May 12

6

15

0 0 0

29 45 36

45 45 45

* Leaves from various positions on extension shoots (terminals): apical leaves, aged I-3 days. had rlol been

sprayed: inlermediate leaves, aged 10-21) days. had been sprayed once t)r twice: and basal leave,,. ~ged 31)-60 days. had been sprayed 3-6 times

Persistence of the fungicides in pear leaves The degradation rate of benomyl in pear leaves was determined by chemical analysis and by bioassay. The chemical analysis, employing a magnesium oxide celite alumina column for clean-up of the sample, converts benomyl, if present, to M BC, and therefore the results are expressed as ppm MBC. The chemical analysis (Fig. 1) showed that the residue level of MBC 4 days after the last benomyl spray (May 12, 1972) was about 50 ppm in the basal leaves. The degradation rate of benomyl

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Phytoparasitica 2:2, December 1974

60

&--~x

BASAL LEAVES

O~O

APICAL LEAVES

20

IO

I

1 40

20

I 60

1 80

I I00

i 120

i 140

I 0---160

I 180

L 200

I 220

DAYS AFTER TREATMENT

Fly. /. Degradation rate of benomyl in pear leaves, as determined by chemical analysis. Leaves from extension shoots (terminals): basals were sprayed seven times, apicals were sprayed once or twice,

I

40

I

I

1

1

1

~...~......~ ~/~ ~

1

1

I

I

I

~ - - - n BASAL LEAVES o o APICAL LEAVES

-4

W ~0--0 Z 0 N Z 0

~m

Z

I0

A o o P., o

o o

=

Benomyl

.

Corbendozim Melhylthiophonotl

I 40

l

I so DAYS

1 AFTER

i ~zo

~

~i~

,

-, ~

=1 "

TREATMENT

Fig 2. Bioassay of pear leaves collected after the last spray in the orchard (May 14, 1972). Leaves from extension shoots (terminals): basals were sprayed seven times and apicals were sprayed once or twice, with benomyl, carbendazim or methylthiophanate.

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during the summer and autumn was very slow and the residue level seven months after treatment was 8 ppm in the basal leaves. Residue levels of MBC in apical leaves which were sprayed only once or twice were lower than those found in the basal leaves. Bioassays of basal leaves (Fig. 2) showed that in the benomyl, carbendazim and methylthiophanate treatments, fungitoxic activity was detected throughout the season; in the apical leaves fungitoxic activity was detectable for five months after the last treatment. In both types of leaves, the activity was similar for all three fungicides. Fungitoxic activity was also detected in leaves collected from the ground, in autumn. Chemical analyses of basal leaves (Table 2) showed that at the end of the summer, 5-7 months after the last treatment, the residue level of MBC was almost the same in the benomyi- and methylthiophanate-treated leaves; the residue level from the carbendazim treatment was somewhat higher. The concentration of the fungicides applied to the trees, when calculated as MBC (ppm equivalent), was 150 ppm of carbendazim, 99 ppm of benomyl, and 117 ppm of methylthiophanate. TABLE RESIDUES

OF MBC

BENOMYL,

FOUND

IN P E A R

CARBENDAZIM

Days after the lust spray

163 192 214

2 LEAVES*

SPRAYED

WITH

OR METHYLTHIOPHANATE

Residues o[ M B C ( p p m ) Treatment

with

benomyl

carbendazim

methylthiophanate

9.4 9.6 8.0

31.3 23.4 -

5.6 6.9 8.1

* Basal leaves from extension shoots (terminals~ sprayed seven limes between March 13 and May 14, 1972.

Residues in fruits Chemical analysis showed (Fig. 3) that in the benomyl-treated fruits the residue level of MBC (average from healthy and infected fruit) decreased from 1.9 to 0.4 ppm during the first 3 weeks after the last treatment. Much of the reduction was probably due to dilution of the toxicant resulting from the growth of the fruit. The residue level in the fruit after two months was almost the same as that found after 21 days. Fruits from the same trees were divided into infected and healthy fruits and analyzed separately. The trend of MBC residue level indicated higher

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Phytoparasitica 2:2, December 1974

residues in the healthy fruits than in the infected ones. Similar results were obtained from infected and less infected halves of pear fruits (Table 3). TABLE 3 RESIDUE LEVEL OF MBC IN PEAR FRUITS AS RELATED TO DEGREE OF SCAB INFECTION

Sampling date

May May May May May June June

Days after last previous spray

18 18 22 30 30 19 25

Residues of MBC in fruits (ppm) Lightly infected fruits

Heavily infected fruits

2.0 1.6" 2.3 2.4* 2.3 0.6 0.5

1.5 1.4" 1.7 1.7" 0.9 0.2 0. I

1 I 5 2 2 21 58

* Infected fruils were divided into infected and Less infected halves.

3.0

[]

HEALTHY FRUIT

[]

INFECTED FRUIT

2.0 o m

1.0

.v

2

21

:; Flo 58

DAYS AFTER TREATMENT

Fig. 3. Residues of MBC in healthy or scab-infected pear fruits sprayed 13 times with benomyl. The last spray was on May 28 and harvest was on July 25: (Results are the average of the analysis of 2 kg of fruit.)

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Analyses of the peel and pulp from benomyl-treated fruits showed 1.4 ppm MBC in the peel and 0.08 ppm MBC in the pulp. These concentrations, when calculated as actual quantities of fungicide (p,g/g fresh wt.), showed that about 85% of the benomyl was in the peel. In this experiment the peel constituted ca. 25% of the whole fruit weight. Treated pear leaves underwent acid and basic hydrolysis which showed that MBC was not bound to the plant tissue. Acetone extraction was sufficient to remove all the MBC from the plant. In earlier work /2t it was shown that MBC, when applied via the root system to pepper and tomato plants, was partially bound to the leaf tissue. The degradation product of M BC, 2-amino-benzimidazole, was detected in pear leaves in small amounts (less than 5% of total MBC).

DISCUSSION In pear trees the young leaves and the fruit are very susceptible to scab disease. During the early growing season new leaves are continuously emerging and therefore frequent spray applications are needed to protect the new growth. The benzimidazole fungicides, when sprayed on the tree, provide protection to leaves that are present at the time of treatment. There is no translocation of benomyl, carbendazim or methylthiophanate from the treated leaves to the neighboring, newly emerging leaves. Thus, the presence of high levels of benzimidazole derivatives in old pear leaves did not contribute much to disease control in the new leaves (E. Shabi, unpublished results). On the other hand, frequent spray applications resulted in the accumulation of high levels of these fungicides in old leaves, which are not susceptible to scab disease. The rate of degradation of these compounds in the leaves is very slow. In the autumn the oldest leaves still contained about 15% of the amount of the toxicant that was applied in the spring, while the youngest leaves contained very small quantities of MBC. Therefore, the effectiveness of the spring treatments against the production of perithecia on fallen leaves during the winter was low. Moreover, there could be the undesirable effect of M B C causing a reduction of the earthworm population (12). Pear fruits are susceptible to scab disease throughout the growing season and therefore frequent spray applications are needed to protect the growing fruit. The rapid decline in the residue level of MBC during the first 3 weeks after the last treatment was due mainly to the rapid growth of the fruit. It seems that proper and continuous coverage of the whole fruit is very important. This assumption is based on the fact that samples and parts of fruit which were more heavily damaged by the disease, were found

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to contain a lower level of the toxicant. Uneven distribution of the fungicide was not corrected by translocation. Thus, in this case, there is no difference between these systemic fungicides and the n0n-systemic ones: in both cases frequent spray applications are needed. At the early stages it is necessary to protect the new, emerging leaves and at later stages the growing fruit. That is why, among other reasons (such as tolerance in fungi to fungicidesl, alternate spray programs should be used in the orchard for disease control. The use of less persistent fungicides under low disease pressure might be more advantageous than the use of benzimidazoles. REFERENCES I,

2.

3. 4. 5.

6. 7. 8.

9. 10

I[. 12.

Aharonson, N. and Ben-Aziz, A. (1973) Determination of residues of benomy[, its hydrolysis product, and thiabendazole in various crops. J. As~. off. agrie. Chem. 50: 13~0-1 ~ 34, Ben-Aziz, A. and Aharonson, N. (1974) Dynamics of uptake, translocation and disappearante of thiabendazole and methy[-2-benzimidazolecarbamate in pepper and tomato plants. Pestle. Biochem. Phy,~iol. 4: 120-126. Burehill, R. T. (1972) Comparison of fungicides for suppressing ascospora production by Ventm'ia inaequalis (Cke.) Wint. PI. Pathol, 21: 19-22. Burehill, R. T. and Williamson, C. J. (1971) Pear scab. Suppression of ascospores by posbharvest sprays. Rep. E. Mulling Res. Stn 1970: 107. Conner, S. R. and Heuberger, J. W. (1968) Apple scab. V. Effect of late-season applications of fungicides on prevention of perithecial development by Venturia imu'quulis. PI. Dis. Reptr 52: 654-658. Doma, S., Clifford, D. R. and Byrde, R. J. W. ([971) Experiments with some of the newer systemic fungicides on apple and marrow. Pestle. Sci. 2: 197-200. Erwin, D. C. (19731 Systemic fungicides: disease control, translocation and mode of action. A. Rec. Phytopathol. !i: 389~22. Kirby, A. H. M. (1972t Progress towards systemic fungicides. Pest Ahstr. News Suture. 18: 1-33. Shabi, E. 11973~ Pear scab control. Results of 1972. Fungic. Nematic. Tests 28: 37-38. Shabi, E,, Pinkas, Y. and Sold, Z. (1974) Distribution of benzimidazole fungicides following pressure injection of pear trees at several growth stages. Phytopathology 64:963 966. Shabi, E., Rotem, J. and Loebenstein, G. (1973) Physiological races of Venturia pirina on pear. Phytopatholo~y 63: 41-43. Wright, M. A. and Stringer, Y. (1973) The toxicity of thiabendazole, benomyl, methyl benzimidazole-2-yl carbamale and thiophanate-methyl to the earthworm, Lumhricus terrcstri~. Pestle. Sci. 4: 431-432.

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