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Development of Wilt in Mint in Response to Infection By Two Pathotypes of


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Development of Wilt in Mint in Response to Infection By Two Pathotypes of Verticillium dahliae and Co-infection by Pratylenchus penetrans.
Dennis A. Johnson and Gerald S. Santo. Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430 and Prosser, WA 99350, respectively.
ABSTRACT

Johnson, D. A. and Santo, G. 2000. Development of wilt in mint in response to infection by two pathotypes of Verticillium dahliae and co-infection by Pratylenchus penetrans.




Two isolates of Verticillium dahliae in vegetative compatibility group (VCG) 2B and one isolate in VCG 4A were tested for aggressiveness on peppermint, Scotch spearmint and native spearmint alone and in combination with the lesion nematode Pratylenchus penetrans. The VCG 2B isolates were more aggressive than the VCG 4A isolate on all three mint species. Severity of wilt was greatest on peppermint and scotch spearmint with the VCG 2B isolates, and colony forming units (cfu’s) of V. dahliae recovered from infected stems were greater in Scotch spearmint when inoculated with V. dahliae and P. penetrans than when inoculated with VCG 2B alone. Wilt severities and cfu’s in peppermint and Scotch spearmint were not increased when inoculated with VCG 4A and P. penetrans compared to VCG 4A alone. These data indicate that V. dahliae isolates from VCG 2B but not VCG 4A interact synergistically with P. penetrans on Verticillium-susceptible mints. The resistance to verticillium in native spearmint was not affected by the presence of lesion nematodes. ________________________________________________________________________
Verticillium wilt, caused by Verticillium dahliae, is a limiting disease of cultivated peppermint, and Scotch spearmint in North America. A third cultivated mint, native spearmint, is more resistant to wilt and is not as severely damaged by the disease as the other two commercial species. Symptoms of the disease are characterized by chlorosis, stunting, asymmetric development of apical leaves, shortening of internodes, and plant mortality (10). Co-infection by the lesion nematode, Pratylenchus penetrans and V. dahliae in peppermint and Scotch spearmint can enhance disease severity (1,6,8,20). Co-infection of potato by V. dahliae and P. penetrans enhances onset and severity of wilt symptoms (2,3,12,18,19). Although the mechanism of this interaction is not fully understood, it appears the nematode may alter or delay host response to infection and colonization by V. dahliae, rather than just causing root wounds that facilitate entrance of the fungus into the vascular system (3,7).


Green (9) demonstrated that V. dahliae from peppermint was not host specific to the genus Mentha, as once thought (16), and presented evidence for the existence of strains of V. dahliae, which varied in host specificity and aggressiveness (9). Although V. dahliae has a wide host range, isolates differ in aggressiveness on various host species (5,9,13,14, 15,17). Vegetative compatibility analysis and pathogenicity tests of V. dahliae isolates from peppermint, spearmint, and other hosts have shown that isolates belonging to vegetative compatibility group (VCG) 2B are more prevalent in infected mint plants in commercial fields and more aggressive in mint than isolates belonging to VCG 4A and VCG 4B (5). Moreover, VCG 4A of V. dahliae appears to be a host-adapted pathotype of potato in North America in that VCG 4A was isolated more frequently from potato stems and tubers, and was more aggressive on potato than VCG 4B (17). VCG 4A isolates and P. penetrans interact synergistically resulting in severe disease and reduced tuber yields on potato; VCG 4B isolates and P. penetrans did not result in a synergistic infection (2). In a recent survey of V. dahliae isolates from potato in Israel, VCG 4B and VCG 2A were recovered whereas VCG 4A was not (14).
This study investigated the interaction of isolates of V. dahliae from VCG 2B and VCG 4A with P. penetrans, on the severity of Verticillium wilt of peppermint, Scotch spearmint and native spearmint. Pathogenicity of three isolates of V. dahliae waqs evaluated on three mint species by inoculating each with specific fungal isolates alone or in combination with P. penetrans.

Severity of Verticillium wilt and cfu’s of V. dahliae did not differ significantly (P > 0.05) among inoculum levels within the first and second experiments, thus data for inoculum level within an experiment were combined and only one inoculum level was used for the third experiment (Table 1). Mean values for wilt severity among mint species and treatments increased between the first and second growth. Because treatment effects were the same for both disease assessments within an experiment, data from the second assessment are presented for the three experiments (Table 1).

Wilt symptoms were greater (P < 0.05) in Black Mitcham peppermint and native spearmint in the first experiment and in Black Mitcham peppermint and Scotch spearmint in the second and third experiments when inoculated with the two VCG 2B isolates as compared with the VCG 4A isolate (Table 1). Numbers of cfu’s of V. dahliae in stems were greater (P < 0.05) in peppermint in the three experiments when inoculated with the VCG 2B isolates than with the VCG 4A isolate (Table 1). Numbers of cfu’s obtained from native spearmint were low and no difference (P > 0.05) between VCG 2B and VCG 4A isolates was observed. Numbers of cfu’s in Scotch spearmint were higher for the VCG 2B isolates than the VCG 4A isolate when in the presence of P. penetrans, but were similar when plants were inoculated only with V. dahliae (Table 1).

Wilt severity and number of cfu’s from plant stems were greater (P < 0.05) for Black Mitcham peppermint than for native spearmint in the first experiment and greater for Black Mitcham peppermint than for Scotch spearmint in the second and third experiments with the VCG 2B isolates, but not with the VCG 4A isolate (Table 1).




In the first experiment, wilt severity and numbers of cfu’s in Black Mitcham peppermint and native spearmint were not increased (P > 0.05) when inoculated with V. dahliae isolates and P. penetrans compared to V. dahliae alone (Table 1). In the second and third experiments, wilt severity was more severe (P < 0.05) in Scotch spearmint when inoculated with the VCG 2B isolates of V. dahliae and P. penetrans than when inoculated with VCG 2B alone. Wilt severity was significantly greater for Black Mitcham peppermint when inoculated with one of two VCG 2B isolates and P. penetrans than when with VCG 2B alone in the second experiment and with both VCG 2B isolates and P. penetrans than with VCG 2B isolates alone in the third experiment. Numbers of V. dahliae cfu’s in stems were significantly higher (P < 0.05) in Scotch spearmint when inoculated with the VCG 2B isolates and P. penetrans than when inoculated with VCG 2B alone. Numbers of cfu’s did not differ significantly (P> 0.05) in Black Mitcham peppermint when inoculated with the VCG 2B isolates and P. penetrans than when inoculated with VCG 2B alone (Table 1). Wilt severity and cfu’s in Black Mitcham peppermint and Scotch spearmint were not increased (P > 0.05) when inoculated with VCG 4A and P. penetrans compared to VCG 4A alone (Table 1).
In all experiments, disease symptoms did not develop on control plants that were not inoculated with either pathogen or those inoculated only with P. penetrans. Mean level of P. penetrans per g dry root weight of plants inoculated with both pathogens was 1085 for Black Mitcham peppermint and 451 for Native spearmint in the first experiment, 1411 for Black Mitcham peppermint and 1230 for Scotch spearmint in the second experiment and 837 for Black Mitcham peppermint and 1834 for Scotch spearmint in the third experiment. P. penetrans was not recovered from plants inoculated only with V. dahliae and from plants not inoculated with either pathogen.

DISCUSSION

A principle finding in this study was that the VCG 2B isolates were capable of interacting with P. penetrans on Black Mitcham peppermint and Scotch spearmint causing increased disease severity whereas the VCG 4A isolate did not. A similar but opposite finding was reported on potato where isolates from VCG 4A interacted with P. penetrans to increase wilt severity, whereas, isolates from VCG 4B did not interact with P. penetrans or did so only weakly (2). On potato, the interaction of V. dahliae and lesion nematodes also varies with the species of Pratylenchus involved (18). A possible explanation for these findings is that P. penetrans affects the host defense mechanisms, allowing the most aggressive strains of V. dahliae to colonize and reproduce (3,7).



The two isolates of V. dahliae from VCG 2B were more aggressive to mint than an isolate from VCG 4A. Wilt severities and numbers of cfu’s were higher in plants inoculated with VCG 2B isolates. The three mint species, with Black Mitcham peppermint being the most susceptible and Native spearmint the most resistant (13), were each more severely affected by the VCG 2B isolates. The relative levels of resistance between Native and Scotch spearmint cannot be compared in this study because inoculum levels differed in the two experiments; however, native spearmint has been demonstrated to be less susceptible than Scotch spearmint (13).

The interaction of a specific VCG of V. dahliae with P. penetrans on either mint or potato supplies additional evidence that VCG 4A is a host-adapted pathotype on potato in North America (17) and VCG 2B is a pathotype on mint (5). We consider a pathotype to be a dominant (frequency of isolation) and highly aggressive strain of a pathogen on a particular host. Other strains of the same pathogenic species may also infect the same host. Supportive evidence for a pathotype concept was originally derived when both VCG 2B and VCG 4A were found to be more prevalent and aggressive on their respective hosts (5,17,21,22). Only a few isolates of V. dahliae were used in this study, but this demonstration of increased aggressiveness on mint by isolates from VCG 2B is consistent with recent studies (5,13). This and previous work (2) demonstrated that the association of P. penetrans with V. dahliae is related to host-adapted strains of V. dahliae. In mint and potato, the association is specific to V. dahliae strain, P. penetrans, and the host.




The resistance in native spearmint in this study was effective against the co-inoculation of both V. dahliae and P. penetrans and no synergistic effect due to the presence of both pathogens was observed. This agreed with earlier studies (Santo, unpublished data). Numbers of cfu’s were low from stems of inoculated native spearmint because of effective host resistance (11,13). In the first experiment a significant increase in disease severity was not observed on Black Mitcham peppermint co-inoculated with the VCG 2B isolates and P. penetrans as compared with V. dahliae alone possibly due to the higher range of V. dahliae inoculum used. The high numbers of microsclerotia used to infest soils in the test may have overwhelmed potential effects due to the nematode (Santo, unpublished data). The intent of the first experiment was to provide sufficient inoculum of V. dahliae so that plant escapes of native spearmint would not be a factor. This was accomplished. Inoculum levels used in these studies produced severe disease symptoms and were adequate for separating effects of the two pathotypes and levels of host resistance.
Management of Verticillium wilt of mint depends on managing the VCG 2B pathotype of V. dahliae and the root-lesion nematode, P. penetrans. The most aggressive isolates of V. dahliae should be used when screening material for resistance (13), and rhizomes used from planting material should be free of VCG 2B. Differentiating among pathotypes of V. dahliae in soil, as well as levels of P. penetrans, may be beneficial in planning management strategies before planting.
ACKNOWLEDGMENTS

This research was supported, in part, by the Mint Industry Research Council. We thank R. C. Rowe for providing the reference strains of the V. dahliae VCG’s, and R. Green, R. C. Rowe, L. Douhan and two anonymous reviewers for critical reviews of the manuscript. PPNS No. 0305, Project 0678, Department of Plant Pathology, College of Agriculture and Home Economics Research Center, Pullman, WA.

LITERATURE CITED

1. Bergeson, G. B. 1963. Influence of Pratylenchus penetrans alone and in combination with Verticillium albo-atrum on growth of peppermint. Phytopathology 53:1164-1166.


2. Botseas, D. D., and Rowe, R. C. 1994. Development of potato early dying in response to infection by two pathotypes of Verticillium dahliae and co-infection by Pratylenchus penetrans. Phytopathology 84:275-282.
3. Bowers, J. H., Nameth, S. T., Riedel, R.,M., and Rowe, R. C. 1996. Infection and colonization of potato roots by Verticillium dahliae as affected by Pratylenchus penetrans and P. crenatus. Phytopathology 86:614-621.
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6. Faulkner, L. R., and Bolander, W. J. 1969. Interaction of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint: Effect of soil temperature. Phytopathology 59: 868-870.
7. Faulkner, L R., Bolander, W. J., and Skotland, C. B. 1970. Interaction of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint: Influence of the nematode as determined by a double root technique. Phytopathology 60: 100-103.
8. Faulkner, L. R., and Skotland, C. B. 1965. Interactions of Verticillium dahliae and Pratylenchus minyus in Verticillium wilt of peppermint. Phytopathology 55:583-586.
9.Green, R. J. 1951. Studies on the host range of the Verticillium that causes wilt of Mentha piperita L. Science 113: 207-208.
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11 Hoyos, G. P., Zambino, P. J., and Anderson, N. A. 1991. An assay to quantify vascular colonization of potato by Verticillium dahliae. Am. Potato J. 68;727-742.
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13. Johnson, D. A., and Cummings, T. F. 2000. Evaluation of mint mutants, hybrids and fertile clones for resistance to Verticillium dahliae. Plant Dis. 84:235-238.
14. Korolev, N., Katan, J., and Katan, T. 2000. Vegetative compatibility groups of Verticillium dahliae in Israel: Their distribution and association with pathogenicity. Phytopathology 90:529-536.
15. Lacy, M. L., and Horner, C. E. 1965. Verticillium wilt of mint: Interactions of inoculum density and host resistance. Phytopathology 55:1176-1178.
16. Nelson, R. 1950. Verticillium wilt of peppermint (Mentha piperita L.). Michigan Agric. Exp. Stn. Tech. Bull. 221. 259pp.
17. Omer, M A., Johnson, D. A., and Rowe, R. C. 2000. Recovery of Verticillium dahliae from N. American certified seed potatoes and characterization of strains by vegetative compatibility and aggressiveness. Amer Jour Potato Res. 77:325-331.
18. Riedel, R. M., Rowe, R. C., and Martin, M. J. 1985. Differential interactions of Pratylenchus crenatus, P. penetrans, and P. scribneri with Verticillium dahliae in potato early dying disease. Phytopathology 75:419-422.


19. Rowe, R. C., Riedel, R. M., and Martin, M. J. 1985. Synergistic interactions between Verticillium dahliae and Pratylenchus penetrans in potato early dying disease. Phytopathology 75:412-418.
20. Santo, G. S., and Skotland, C. B. 1986. Interaction of Pratylenchus penetrans and Verticillium dahliae on Scotch spearmint in microplots. Jour Nematol. 18:631.
21. Strausbaugh, C. A. 1993. Assessment of vegetative compatibility and virulence of

Verticillium dahliae isolates from Idaho potatoes and tester strains. Phytopathology

83:1253-1258.
22. Strausbaugh, C. A., Schroth, M N., Weinhold, A. R. and Hancock. J. G. 1992. Assessment of vegetative compatibility of Verticillium dahliae tester strains and isolates from California potatoes. Phytopathology 82:61-68.
Table 1. Severity of wilt symptoms and colony forming units of Verticillium dahliae from stems of Black Mitcham peppermint, Native spearmint and Scotch spearmint inoculated with three isolates in two different vegetative compatibility groups alone and in combination with Pratylenchus penetransa





Mint Species/Isolate


Wilt Severity Indexb


Colony Forming Units

(cfu/cm stem)




First Experimentc








Black Mitcham




VCG


V.d.d


V.d. + P.p.e


V.d.


V.d. + P.p.

Isolate 50


2B

4.1

4.2

369

498

Isolate 109

2B

3.9

4.0

131

198

Isolate 653

4A



2.3*


2.7*


0*


73*

Native Spearmint












Isolate 50


2B

3.6

3.5

0

0

Isolate 109

2B

3.0

3.2

22

3.6

Isolate 653

4A

2.5*



2.7*


0


0














Second Experimentc










Black Mitcham












Isolate 50


2B

4.0

4.0

39

20

Isolate 109

2B

4.1

4.5t


117

267

Isolate 653


4A



1.9*


2.1*


0.04*


0.04*

Scotch Spearmint












Isolate 50


2B

2.8

3.2t


0.6

16t

Isolate 109


2B

2.6

3.3t


0.4

13t

Isolate 653


4A



2.3*


2.1*


2.0


0.04*


Third Experimentc










Black Mitcham












Isolate 50


2B

2.8

3.2t


238

154

Isolate 109


2B

3.4

3.8t


282

248

Isolate 653


4A

1.3*

1.6*

0*

0*

Scotch Spearmint











Isolate 50


2B

2.1

3.3t


0

7t

Isolate 109


2B

2.2

3.4t


0

42t

Isolate 653


4A

1.2*

1.3*

0

0*




a Values are means of nine replicates.

b Wilt severity Index: 0 = healthy, 5 > 40% necrotic tissue.

c Inoculum concentration levels were 100, 200, and 300 microsclerotia/cm3 soil in the first experiment, 30 and 60 microsclerotia/cm3 soil in the second experiment and 30 microsclerotia/cm3 soil in the third experiment. Wilt severity and cfu’s did not differ significantly among inoculum levels within each experiment; thus data for inoculum levels within an experiment were combined.

d Verticillium dahliae

e Pratylenchus penetrans

t Significantly greater (P < 0.05) when co-inoculated than when only inoculated with V. dahliae.

* Value for isolate 653 (VCG 4A) is significantly less (P < 0.05) than values for VCG 2B isolates.

** Significantly greater (P = 0.05) when co-inoculated than only inoculated with V. dahliae.

Dennis A. Johnson, Page , Plant Disease




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