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Carbendazim exudation from roots of American elm
SHORT COMMUNICATION Carbendazim exudation from roots of American elm WILLIAM H. SMITH and DOUGLASM. HAEFELE
School of Forestry and Environmental Studies, Yale University, New Haven, CT 06511, U.S.A. (Accepted
20 June 1979)
The use of the systemic fungicide benzimidazolecarbamate for the control of woody plant diseases is receiving considerable attention (Erwin, 1973; Marsh, 1977). Stem injec-’ tion of benomyf [m~thyI-l~butyIcarbamoyl)-2-benzimidazol~arbamate] and MBC [methyl-2-~nzimidazole~rbamate] formulations have been suggested for control of Dutch elm disease, chestnut blight, oak wilt and pear scab. Following trunk injection of benomyl or MBC, carhendazim is translocated primarily via the xylem to the leaves and twigs of the crown. Some translocation may also occur downward via the phloem to the roots of injected trees (Jaynes and Van Allen, 1977; Prasad and Travnick, 1973; Travnick and Prasad, 1974). The potential for movement into the soil via root exudation, raises the possibility of non-target effects on mycorrhizal symbionts as well as rhizosphere microbes. In a study employing [‘4C]-benomyl and American elm seedlings, some “excretion” of labeled C was observed (R. Prasad, personal communication; Prasad and Travnick, 1974). Our purpose was to determine the presence of carbendazim in the root exudates of Ufmus a~r~eu~a t. saplings following “commercial dose” injection of Lignasan BLP under field conditions. An unfloored 3 x 3 m metal garden storage building was erected on a 5 x 5 cm wooden support frame in our tree nursery. The central area under the building had been excavated to a depth of 40 cm. In April 1976, two U. americam saplings (Princeton Nurseries, Dedfree patented clone, 7-10yr old) were planted 45 cm from each of the three non-door sides of the building. Before planting holes with l-m wide channels under the building walls had been excavated and back-filled with a greenhouse soil mixture. During the 1976 growing season the trees were pruned and watered and they grew vigorously. Excellent root development occurred into the building. In May 1977, all trees Table 1. Occurrence of carbendazim
Tree
(ml
Dia ~1 14m (lXIl)
I
40
35
2.ot 1.0:
Height
Carbendarim PO, mjection dose &cm-’ dza)
were growing well and ranged in height from 3.4 to 4.0m and in diameter at 1.4 m from 3.2 to 3.8 cm. In mid-May 1977 portions of the root systems of the six experimental trees were prepared for root exudate collection (Smith, 1970). On 13 June 1977 four trees were pressure injected (2 bars) with 0.8% carbendazimphosphate (Lignasan BLP, methyl 2-benzimidazolecarbamate phosphate) through two l-cm deep holes (5 mm dia) drilled in opposite sides of the trees 5 cm above the soil line (Jaynes and Van Alfen, 1977). Two trees were injected with 1.0 g active ingredient cm-i dia (Connecticut commercial injection rate) and two trees were injected with 2.0 g active ingredient cm-’ dia. Two control trees were injected with distilled water. Root exudates were collected at 48-65 h, 24 days and 89 days after injection. The presence of carbendazim in root exudates was determined by thin layer chromatography on silica gel plates (Balinova, 1975; Sherma, 1975). Considerable variation occurred in the uptake of Lignasan BLP by injected trees. The uptake was also relatively slow and averaged 18 h. Injection was terminated when all the material was taken up or at 29 h. A Penicillium bioassay indicated good carbendazim distribution to the uppermost leaves and twigs at 48 h. Root sample bioassays at 48 h proved negative. The injection of Lignasan BLP resulted in phytotoxicity including wilt, defoliation and scorch on all treated trees, however, all symptoms were partial and none persisted beyond 4 weeks (Table 1). All elms injected with Lignasan BLP released carbendazim in their root exudate. The positive entries of Table 1 represent consistent appearance of carbendazim spots as detected by reagent spray and U.V. quenching by the two development procedures. The amounts released were small and the TLC procedures employed proved to be only
in the rbot exudate of American elm saplings following pressure injection of carbendazim phosphate (Lignasan BLP)
Carbendaztm PO, actuai uprake gem-’ g itcAaii 19
6.8
Phy~o~oxici~y symptoms* Wilt Derollatlon Wilt. scorch Defoliatmn
mg 277
Dry wt or roots yielding. exudates and occurrence 0.l.c.) olcarbendazim in exudate Time following injection 48-65 h 89 days 24 days Carbendanm mg Carbendarim mg Carbendazim a
256
POSltlVe
174
Powive
2
40
3.5
0.9
3.0
234
0
269
POSlliVe
276
POSlllVe
3 4 5
3.7 34 3.4
3.x 3.2 3.5
I .o: 2ot 0 (control)
0.7 0.7 0
2.8 2.1 0
Delol~atmn Defohation None
555 377 Ml
Positive\ 0 0
375 336 331
0 POSltiVe Positrve
I86 199 301
Positive 0 0
6
4.0
3.5
Icontrol)
0
0
NolIe
333
0
223
0
246
0
* Appeared three days following injection and were all partial and of minor significance; no symptoms persisted beyond 4 weeks. t Two times Connecticut rate of commercial injection. t: Connecticut rate of commercial injection. 5 More than zero, but less than 2 mg.
687
688
Short communications
qualitative. One of the control trees yielded a positive carbendazim test at 24 days. Given the small number of trees injected, the sapling size of the experimental trees. and the occasionally irregular movement of systemic materials the variable nature of carbendazim detection in the exudates is not surprising. The only tree to exhibit carbendazim in the first sample period (48-65 h) probably did so because of the relatively large root volume (555 mg) produced in the collection tubes. The failure of this same tree to release carbendazim at 24 days is peculiar and unexplained. Likewise unclear is the failure of tree 4 to release at 89 days after detection at 24 days. ~tlthough the root mass at 89 days was relatively small compared to the previous collections. The carbendazim reported in the exudate of a control tree in 24 days is thought to have been due to root grafting or proximate root transfer. A less likely explanation is the possibility that carbendazim leached from defoliated leaves may have entered the soil solution, been absorbed by control tree roots and released in exudates. Two precautions were taken to avoid inadvertent transfer immediately after the 24-day collection. All defoliated leaves were removed from the test area and 90 x 120cm plexiglass sheets (7 mm thick) were inserted in trenches dug between adjacent trees. None of the control trees released carbendazim at 89 days. While the amount of carbendazim released in root exudates was small, it was consistent. If assumed that a positive detection was equivalent to approximately 1 pg (threshold of detection by our procedures). and that this was obtained from roots weighing l74mg (smallest root mass--tree 1, 89 days) then the maximum release under the conditions of this experiment may have approximated 5 ng mg- ’ dry root. The specific chemistry of carbendazim released is not known. In aqueous solution, carbendazim may exist in the cationic, anionic or unionized form. Preferential uptake of the molecular form has been documented with excised corn roots (Leroux and Gredt, 1975). If the mechanism of root exudate loss is passive, the form of exuded carbendazim may well be molecular. Incorporation of benomyl in soil samples has resulted in variable effects on the non-target soil microhora (Hofer et al., 1971; Peeples, 1974; Ponchet and Tramier, 1971; Siegel, 197.5). None of these studies, however, is directly comparable to the rhizosphere situation. Reduction of earthworm populations in forest soils treated with benomyl and MBC has been documented [Moody and Prasad, 1974; Webb and Newton, 1972). Carbendazim should be added to the list (Foy et al., 1971; Webb and Newton, 1972) of pesticides that may be introduced into the rhizosphere via root exudation. Future research should provide more quantitative results on release, examine larger trees. and determine specific effects on important rhizosphere organisms.
A~knowlr~~~ments-This research was supported by a grant from the National Science Foundation. Gerald Walton, Connecticut Agricultural Experiment Station, assisted with tree injection.
BALINOVA A. (1975) Thin-layer chromatographic detection of some systemic fungicides and their metabolites. Journul of ~hromurugruph~ 1 I I, I97 199. ERWIN D. C. (1973) Systemic fungicides: Disease control, translocation, and mode of action. Atmuul Review c$’ Phytopathology 11,389422. Fou C. L.. HURTT W. and HALE M. G. (1971) Root exudation of plant-growth regulators, In Biochemical Inwracrions Among Plunts, pp. 7585. National Academy of Sciences, Washington, DC. HOFER V. I.. BETH T. and WALLN~FER P. (1971) Der Einfluss des Fungizids Benomyl auf die Bodermikroflora.
Zritschr$tfiir
I’jfunzenkrunkheiren
und
Pfiunzenschurz
78, 398405.
JAYNES R. A. and VAN ALFEN N. K. (1974) Control of American chestnut blight by trunk injection with methyl2-benzimidazol carbamate (MBC). Phytopathology 46, 1479-1480.
JAYNES R. A. and VAN ALFEN N. (1977) Control of the chestnut blight fungus with injected methyl-l-t-benzimidazolecarbamate. Plnnr Disease Reporrer 61, 1032~1036. LER~UX P. and GREI)T M. (1975) Absorption of methyl benzimidazol-2-yl carbamate (carbendazim) by corn roots. Pesticide Biochemistry und Physioloy,y 5, 507.. 5 14. MARSH R. W. (Ed.) (1977) Swemic Fungicides. 401 pp. Longman, New York. MWDY R. P. and PRASAD R. (1974) Impact of pesticides on forest ecosystems. Reduction of earthworm populations in forest soils following treatment of elm trees with benomyl. Proceedings Canadiun Frderotion of’ Biologic,u/ Societies (Hamilron) 17, 100. PEEPLES J. L. (1974) Microbial activity in benomyl-treated soiis. P~~rop~fr~u~og~~ 64, 857-860. PONCAET J. and TRAMIER R. (1971) Effects du benomyl sur la croissance de l’oeillet et al microflore des sols traites. Annals of Phytopathology
3, 401406.
PRASAD R. and TRAVNICK D. (1973) Translocation of henomyl in elm (Ulmus umericana L.)_.---V. Distribution patterns in mature trees following trunk-injection under high pressures. Chemical Control Research Institute, Information Report CC-X-53, Ottawa, Ontario, 28 pp. PRASAD R. and TRAVNI~K D. (1974) Fate of pesticides in forest trees. Transport, accumulation and excretion of benomyl by UImus americana L. Proceedings Canadian Federation of Biologicul Societies (Hamilton) 17, 100. SWERMA J. (1975) Quantification of benomyl and its metabolites by thin layer densitometry. .lourna/ of Chromatography
104,47&479.
SIEGEL M. R. (1975) Benomyl-soil microbial interactions. P~~rop~i~o~~g.v 65, 2 i 9-220. SMITH W. H. (1970) Technique for collection of root exudates from mature trees. Plant & Soil 32, 2388241. TRAVNICK D. and PRASAD R. (1974) Translocation of benomyl (MBC-chloride) and rhodamine dye in Ulmus americunu L. following stem-injection under high pressures. Federation of’ Biologictrl Societies (Hamilton, Cunadu)
17, 102.
WERB W. L. and NEWX~N M. (1972) Release from roots. Weed Research 12, 391-394.
of picloram
School of Forestry and Environmental Studies, Yale University, New Haven, CT 06511, U.S.A. (Accepted
20 June 1979)
The use of the systemic fungicide benzimidazolecarbamate for the control of woody plant diseases is receiving considerable attention (Erwin, 1973; Marsh, 1977). Stem injec-’ tion of benomyf [m~thyI-l~butyIcarbamoyl)-2-benzimidazol~arbamate] and MBC [methyl-2-~nzimidazole~rbamate] formulations have been suggested for control of Dutch elm disease, chestnut blight, oak wilt and pear scab. Following trunk injection of benomyl or MBC, carhendazim is translocated primarily via the xylem to the leaves and twigs of the crown. Some translocation may also occur downward via the phloem to the roots of injected trees (Jaynes and Van Allen, 1977; Prasad and Travnick, 1973; Travnick and Prasad, 1974). The potential for movement into the soil via root exudation, raises the possibility of non-target effects on mycorrhizal symbionts as well as rhizosphere microbes. In a study employing [‘4C]-benomyl and American elm seedlings, some “excretion” of labeled C was observed (R. Prasad, personal communication; Prasad and Travnick, 1974). Our purpose was to determine the presence of carbendazim in the root exudates of Ufmus a~r~eu~a t. saplings following “commercial dose” injection of Lignasan BLP under field conditions. An unfloored 3 x 3 m metal garden storage building was erected on a 5 x 5 cm wooden support frame in our tree nursery. The central area under the building had been excavated to a depth of 40 cm. In April 1976, two U. americam saplings (Princeton Nurseries, Dedfree patented clone, 7-10yr old) were planted 45 cm from each of the three non-door sides of the building. Before planting holes with l-m wide channels under the building walls had been excavated and back-filled with a greenhouse soil mixture. During the 1976 growing season the trees were pruned and watered and they grew vigorously. Excellent root development occurred into the building. In May 1977, all trees Table 1. Occurrence of carbendazim
Tree
(ml
Dia ~1 14m (lXIl)
I
40
35
2.ot 1.0:
Height
Carbendarim PO, mjection dose &cm-’ dza)
were growing well and ranged in height from 3.4 to 4.0m and in diameter at 1.4 m from 3.2 to 3.8 cm. In mid-May 1977 portions of the root systems of the six experimental trees were prepared for root exudate collection (Smith, 1970). On 13 June 1977 four trees were pressure injected (2 bars) with 0.8% carbendazimphosphate (Lignasan BLP, methyl 2-benzimidazolecarbamate phosphate) through two l-cm deep holes (5 mm dia) drilled in opposite sides of the trees 5 cm above the soil line (Jaynes and Van Alfen, 1977). Two trees were injected with 1.0 g active ingredient cm-i dia (Connecticut commercial injection rate) and two trees were injected with 2.0 g active ingredient cm-’ dia. Two control trees were injected with distilled water. Root exudates were collected at 48-65 h, 24 days and 89 days after injection. The presence of carbendazim in root exudates was determined by thin layer chromatography on silica gel plates (Balinova, 1975; Sherma, 1975). Considerable variation occurred in the uptake of Lignasan BLP by injected trees. The uptake was also relatively slow and averaged 18 h. Injection was terminated when all the material was taken up or at 29 h. A Penicillium bioassay indicated good carbendazim distribution to the uppermost leaves and twigs at 48 h. Root sample bioassays at 48 h proved negative. The injection of Lignasan BLP resulted in phytotoxicity including wilt, defoliation and scorch on all treated trees, however, all symptoms were partial and none persisted beyond 4 weeks (Table 1). All elms injected with Lignasan BLP released carbendazim in their root exudate. The positive entries of Table 1 represent consistent appearance of carbendazim spots as detected by reagent spray and U.V. quenching by the two development procedures. The amounts released were small and the TLC procedures employed proved to be only
in the rbot exudate of American elm saplings following pressure injection of carbendazim phosphate (Lignasan BLP)
Carbendaztm PO, actuai uprake gem-’ g itcAaii 19
6.8
Phy~o~oxici~y symptoms* Wilt Derollatlon Wilt. scorch Defoliatmn
mg 277
Dry wt or roots yielding. exudates and occurrence 0.l.c.) olcarbendazim in exudate Time following injection 48-65 h 89 days 24 days Carbendanm mg Carbendarim mg Carbendazim a
256
POSltlVe
174
Powive
2
40
3.5
0.9
3.0
234
0
269
POSlliVe
276
POSlllVe
3 4 5
3.7 34 3.4
3.x 3.2 3.5
I .o: 2ot 0 (control)
0.7 0.7 0
2.8 2.1 0
Delol~atmn Defohation None
555 377 Ml
Positive\ 0 0
375 336 331
0 POSltiVe Positrve
I86 199 301
Positive 0 0
6
4.0
3.5
Icontrol)
0
0
NolIe
333
0
223
0
246
0
* Appeared three days following injection and were all partial and of minor significance; no symptoms persisted beyond 4 weeks. t Two times Connecticut rate of commercial injection. t: Connecticut rate of commercial injection. 5 More than zero, but less than 2 mg.
687
688
Short communications
qualitative. One of the control trees yielded a positive carbendazim test at 24 days. Given the small number of trees injected, the sapling size of the experimental trees. and the occasionally irregular movement of systemic materials the variable nature of carbendazim detection in the exudates is not surprising. The only tree to exhibit carbendazim in the first sample period (48-65 h) probably did so because of the relatively large root volume (555 mg) produced in the collection tubes. The failure of this same tree to release carbendazim at 24 days is peculiar and unexplained. Likewise unclear is the failure of tree 4 to release at 89 days after detection at 24 days. ~tlthough the root mass at 89 days was relatively small compared to the previous collections. The carbendazim reported in the exudate of a control tree in 24 days is thought to have been due to root grafting or proximate root transfer. A less likely explanation is the possibility that carbendazim leached from defoliated leaves may have entered the soil solution, been absorbed by control tree roots and released in exudates. Two precautions were taken to avoid inadvertent transfer immediately after the 24-day collection. All defoliated leaves were removed from the test area and 90 x 120cm plexiglass sheets (7 mm thick) were inserted in trenches dug between adjacent trees. None of the control trees released carbendazim at 89 days. While the amount of carbendazim released in root exudates was small, it was consistent. If assumed that a positive detection was equivalent to approximately 1 pg (threshold of detection by our procedures). and that this was obtained from roots weighing l74mg (smallest root mass--tree 1, 89 days) then the maximum release under the conditions of this experiment may have approximated 5 ng mg- ’ dry root. The specific chemistry of carbendazim released is not known. In aqueous solution, carbendazim may exist in the cationic, anionic or unionized form. Preferential uptake of the molecular form has been documented with excised corn roots (Leroux and Gredt, 1975). If the mechanism of root exudate loss is passive, the form of exuded carbendazim may well be molecular. Incorporation of benomyl in soil samples has resulted in variable effects on the non-target soil microhora (Hofer et al., 1971; Peeples, 1974; Ponchet and Tramier, 1971; Siegel, 197.5). None of these studies, however, is directly comparable to the rhizosphere situation. Reduction of earthworm populations in forest soils treated with benomyl and MBC has been documented [Moody and Prasad, 1974; Webb and Newton, 1972). Carbendazim should be added to the list (Foy et al., 1971; Webb and Newton, 1972) of pesticides that may be introduced into the rhizosphere via root exudation. Future research should provide more quantitative results on release, examine larger trees. and determine specific effects on important rhizosphere organisms.
A~knowlr~~~ments-This research was supported by a grant from the National Science Foundation. Gerald Walton, Connecticut Agricultural Experiment Station, assisted with tree injection.
BALINOVA A. (1975) Thin-layer chromatographic detection of some systemic fungicides and their metabolites. Journul of ~hromurugruph~ 1 I I, I97 199. ERWIN D. C. (1973) Systemic fungicides: Disease control, translocation, and mode of action. Atmuul Review c$’ Phytopathology 11,389422. Fou C. L.. HURTT W. and HALE M. G. (1971) Root exudation of plant-growth regulators, In Biochemical Inwracrions Among Plunts, pp. 7585. National Academy of Sciences, Washington, DC. HOFER V. I.. BETH T. and WALLN~FER P. (1971) Der Einfluss des Fungizids Benomyl auf die Bodermikroflora.
Zritschr$tfiir
I’jfunzenkrunkheiren
und
Pfiunzenschurz
78, 398405.
JAYNES R. A. and VAN ALFEN N. K. (1974) Control of American chestnut blight by trunk injection with methyl2-benzimidazol carbamate (MBC). Phytopathology 46, 1479-1480.
JAYNES R. A. and VAN ALFEN N. (1977) Control of the chestnut blight fungus with injected methyl-l-t-benzimidazolecarbamate. Plnnr Disease Reporrer 61, 1032~1036. LER~UX P. and GREI)T M. (1975) Absorption of methyl benzimidazol-2-yl carbamate (carbendazim) by corn roots. Pesticide Biochemistry und Physioloy,y 5, 507.. 5 14. MARSH R. W. (Ed.) (1977) Swemic Fungicides. 401 pp. Longman, New York. MWDY R. P. and PRASAD R. (1974) Impact of pesticides on forest ecosystems. Reduction of earthworm populations in forest soils following treatment of elm trees with benomyl. Proceedings Canadiun Frderotion of’ Biologic,u/ Societies (Hamilron) 17, 100. PEEPLES J. L. (1974) Microbial activity in benomyl-treated soiis. P~~rop~fr~u~og~~ 64, 857-860. PONCAET J. and TRAMIER R. (1971) Effects du benomyl sur la croissance de l’oeillet et al microflore des sols traites. Annals of Phytopathology
3, 401406.
PRASAD R. and TRAVNICK D. (1973) Translocation of henomyl in elm (Ulmus umericana L.)_.---V. Distribution patterns in mature trees following trunk-injection under high pressures. Chemical Control Research Institute, Information Report CC-X-53, Ottawa, Ontario, 28 pp. PRASAD R. and TRAVNI~K D. (1974) Fate of pesticides in forest trees. Transport, accumulation and excretion of benomyl by UImus americana L. Proceedings Canadian Federation of Biologicul Societies (Hamilton) 17, 100. SWERMA J. (1975) Quantification of benomyl and its metabolites by thin layer densitometry. .lourna/ of Chromatography
104,47&479.
SIEGEL M. R. (1975) Benomyl-soil microbial interactions. P~~rop~i~o~~g.v 65, 2 i 9-220. SMITH W. H. (1970) Technique for collection of root exudates from mature trees. Plant & Soil 32, 2388241. TRAVNICK D. and PRASAD R. (1974) Translocation of benomyl (MBC-chloride) and rhodamine dye in Ulmus americunu L. following stem-injection under high pressures. Federation of’ Biologictrl Societies (Hamilton, Cunadu)
17, 102.
WERB W. L. and NEWX~N M. (1972) Release from roots. Weed Research 12, 391-394.
of picloram