Differential susceptibility of Russian thistle accessions to Colletotrichum gloeosporioides

William Bruckart , , a, Craig Cavina, Laszlo Vajnab, Ildiko Schwarczingerb and Frederick J. Ryanc a USDA-ARS-FDWSRU, 1301 Ditto Ave., Ft. Detrick, MD 21702, USA b Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, Budapest 1525, Hungary c USDA-ARS-EIDP, 9611 South Riverbend Ave., Parlier, CA 93648, USA Received 16 May 2003; accepted 1 December 2003. Available online 30 December 2003.

Abstract

Molecular information suggests that Russian thistle (Salsola tragus L.) in the US may consist of more than one genetic entity. This genetic variation needs to be taken into account when developing agents for biological control of this important weed. Preliminary evidence suggests that there are differences in susceptibility of Salsola sp. to infection by fungal pathogens. In the present study, an isolate of Colletotrichum gloeosporioides, a pathogen of Russian thistle collected in Hungary was tested for its ability to infect and damage California accessions of S. tragus (referred to as Type A) and the related S. tragus, Type B. The minimum dew period and temperature required for infection of S. tragus with C. gloeosporioides was determined to be 12–16 h and 25 °C. Both Type A and Type B were susceptible, but C. gloeosporioides caused greater damage and reductions in biomass of Type A than of Type B. Fresh weights of Type A and Type B were reduced from controls by 60 and 9%, respectively, after inoculations with C. gloeosporioides. Results from this study illustrate the importance of understanding target plant taxonomy in biological control evaluations.

Author Keywords: Salsola tragus; Salsola australis; Salsola iberica; Salsola kali; Colletotrichum gloeosporioides; Chenopodiaceae; Uredinales; Biological control; Weeds; Rust fungus; Plant taxonomy; Russian thistle; Biotypes

Article Outline

1. Introduction 2. Materials and methods 2.1. The pathogen and inoculation procedures 2.2. Salsola acquisitions tested 2.3. Experiments 2.4. Data collection 2.5. Data analysis 3. Results 3.1. Infection requirements for Colletotrichum gloeosporioides 3.2. Effect of pathogenic fungi on Russian thistle accessions 4. Discussion Acknowledgements References

1. Introduction

Russian thistle, or tumbleweed, is a major weed pest of the US that displaces valuable forage and crop plants, serves as a reservoir for several important vegetable viruses and insects, generates allergenic pollen, injures farm workers and animals, poisons sheep, causes traffic hazards, and raises the potential for wild fires (Crompton and Bassett, 1985; Young and Evans, 1985). Management is difficult, largely because of the relatively high cost of conventional control options in range and pasture agriculture, but also because Russian thistle has developed resistance to the sulfonylurea herbicides in certain agricultural settings ( Saari et al., 1992; Young et al., 1995).

Currently, Russian thistle is classified as one species, Salsola tragus L. Historically, there have been numerous scientific names (as different species, varieties, and subspecies) applied to this plant in North America (Crompton and Bassett, 1985; Mosyakin, 1996; Wilken, 1993), which has caused confusion in documentation of the plant as a pest. Among the names is Salsola kali L., used in the original study on Uromyces salsolae Reichardt for biological control (Hasan et al., 2001) and misapplied by the authors in that study with Salsola pestifer A. Nelson, Salsola australis R. Brown, and Salsola iberica Sennen and Pau. The latter two names also are synonyms of S. tragus (Wilken, 1993). More recently, genetic variability has been investigated within S. tragus in California (Ryan and Ayres, 2000), leading to the conclusion that there were two different genetic entities present there. These were described as “types” of S. tragus. “Type A” in that work is now considered to be the true S. tragus, but it is referred to as Type A in this paper. “Type B” is morphologically similar but genetically distinct and may be a separate species from Type A.

Previous work on biological control agents demonstrated that genetic variation in weeds must be taken into account in the selection of agents (Burdon et al., 1981; Hasan, 1985; Sobhian and Andres, 1978). Two lepidopterous insects (Coleophora klimeschiella Toll and Coleophora parthenica Meyrick) were released for the control of Russian thistle in the 1960s, but they have not been effective (Goeden and Pemberton, 1995; Müller et al., 1990). Recent work has shown that Type A and Type B from California were differentially attacked by a potential biological control agent, the gall midge Desertovellum stackelbergi Mamaev (Diptera: Cecidomyiidae) in field plots in Uzbekistan (Sobhian et al., 2003). In this free-choice situation, the gall midge preferred Type A but it is still under consideration as a biological control agent since it attacked both types.

Two pathogens are under consideration for biological control of Russian thistle in the United States. These are Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. in Penz., a fungus collected in Hungary that causes cankers on stems and leaves (Schwarczinger et al., 1998), and the rust fungus, U. salsolae, from Turkey (Hasan et al., 2001). Results from preliminary inoculation studies indicated variability in susceptibility between different Salsola “species” (identified by collectors) and accessions of “S.tragus” (Bruckart et al., 2000) to each of these pathogens. Objective of this study was to determine the susceptibility of Type A and Type B from California to C. gloeosporioides.

2. Materials and methods

2.1. The pathogen and inoculation procedures

Colletotrichum gloeosporioides isolate 96-067 collected from “S. kali” in Hungary by L. Vajna (Hungarian Academy of Sciences, Budapest) was used in these tests. The isolate was maintained on V-8 Juice agar. Back-up cultures were kept either on half-strength corn meal agar slants in screw-cap tubes or as agar plugs in Nunc vials in liquid nitrogen. Inoculum consisted of a suspension of conidia harvested from 20% V-8 Juice agar cultures after 2-week growth. Plants were inoculated 4 weeks after transplanting by spraying with 106 conidia/ml until the foliage was completely wet. All plants were exposed to 12–16 h dew at 25 °C and then transferred to a greenhouse (20–25 °C) until symptoms developed. Supplemental lighting was provided from October–March to give a 16-h photoperiod.

2.2. Salsola acquisitions tested

Six collections of S. tragus representing two different types (Ryan and Ayres, 2000) were inoculated in these studies ( Table 1). All accessions were from California and identified to type using isozymes (Ryan and Ayres, 2000). Table 1. Collections of Salsola tragus from California used in these studies

2.3. Experiments

Optimal conditions for infection of Type A by C. gloeosporioides in the greenhouse were determined. Plants (5 per treatment combination, 100 total) of Type A were inoculated as described and subjected to dew at different temperatures (10, 15, 20, 25, and 30 °C) and time periods (4, 8, 12, and 16 h). This experiment was run twice. Data were collected and analyzed as described below.

The relative susceptibility of Type A and Type B accessions to infection by was determined. Both Type A and Type B plants were included in each of five replicates (n>500). Plants were inoculated as described, given 16–20 h dew at 25 °C on the basis of results from tests with C. gloeosporioides, and monitored as described.

2.4. Data collection

Disease ratings were made each week for 4 weeks in each experiment and the ratings from the fourth week were used for analysis. Ratings were based on a scale from 0 to 4, where: 0, no macroscopic symptoms; 1, small or isolated lesions, <25% of the plant infected; 2, some coalesced lesions, 25–50% of the plant infected; 3, many coalesced lesions, >50% of the plant infected; and 4, severe infection, dead plant.

At the end of each experiment, plants cut off at the soil line were removed and weighed (= fresh weight, FWT), dried overnight at 100 °C, and weighed again (= dry weight, DWT). A third variable, weight change (WTCH), was calculated to standardize biomass data between individual plants; it was derived by dividing the difference between FWT and DWT by the FWT, i.e., WTCH = (FWT−DWT)/FWT. 2.5. Data analysis

Data for FWT and DWT were transformed to the log10+ 0.1 equivalent (= LFWT and LDWT, respectively) and tested for normality using Proc Univariate in SAS (Statistical Analysis Systems, Cary, NC; v 6.12). LDWT data were subjected to the analysis of variance using Proc GLM in SAS. LFWT data were subjected to the analysis of covariance using Proc GLM and LDWT as the covariate. WTCH data were transformed to the arcsine equivalent before analysis of variance.

Least Squares Means (LSMEANS, SAS), estimated for each treatment combination, were compared on the basis of probabilities that pairs were equivalent (H0: LSMEAN (i)=LSMEAN (j), where treatment ‘i’ is C. gloeosporioides, and treatment ‘j’ is the uninoculated control). Mean separations were made only within type between each treatment and its control. Disease rating data from the study on optimal conditions for infection by C. gloeosporioides were analyzed using Proc NPAR1WAY, the Wilcoxon option, of SAS for dew period, temperature, and treatment. The same analysis was applied by type within treatment to disease rating data on susceptibility of Salsola types.

3. Results

3.1. Infection requirements for Colletotrichum gloeosporioides

Infection and damage of Type A increased with increasing dew temperatures and dew periods after inoculation with C. gloeosporioides (Table 2, Fig. 1A). Fresh weight (FWT) of inoculated plants was less than that of controls (Table 2) after 12 h dew at 25 and 30 °C (P=0.08 and <0.01, respectively), and after 16-h dew at 25 and 30 °C (P 0.01). There were no significant effects associated either with shorter dew periods (data not shown) or with temperatures cooler than 25 °C at 12 or 16 h dew (Table 2). Disease ratings, based on symptomatology, clearly supported results from the effects of infection conditions (dew temperature and dew period) on disease development ( Fig. 1). Similar results were noted also for effects of infection conditions on DWT and WTCH (data not shown).

Display Full Size version of this image (11K)

Fig. 1. Average disease ratings for Salsola tragus (Type A) either inoculated with Colletotrichum gloeosporioides (A) or not [i.e., untreated controls (B)] and incubated at different temperatures (Temperature, °C) for different lengths of time in dew chambers (Dew Period, h). The rating scale is from 0 to 4, where ‘0’ is non-visible disease and ‘4’ is a severe infection and plant death.

Table 2. Mean fresh weights (FWT)a of Salsola tragus (Type A) inoculated with Colletotrichum gloeosporioides (Cg) and provided 12 or 16 h of dew at different temperatures

Uninoculated Type A control plants incubated at 30 °C for 12 or 16 h were visibly damaged (i.e., rated for disease), but there was no evidence of infection by C. gloeosporioides. FWT of these plants after 12 or 16 h of dew was 6.94 and 5.22 g, respectively (Table 2), and were less than pooled means of controls by 15.4 and 31.9%, respectively. Pooled means of controls were calculated by averaging LSMEANS of data from 12 or 16 h dew periods at 10, 15, 20, and 25 °C (Table 2) and were 8.20 g (c.i. ± 0.63, P=0.05) and 7.67 g (c.i. ± 0.88, P=0.05), respectively. Disease ratings also were higher after uninoculated control plants were incubated at 30 °C for 16 h (Fig. 1B).

3.2. Effect of pathogenic fungi on Russian thistle accessions Inoculations with C. gloeosporioides resulted in reductions in the biomass of Russian thistle accessions. Significant differences were noted between treated and control plants for both Type A and Type B, but damage to Type A was much greater than to Type B (Table 3). The variables DWT, WTCH, and FWT of Type A inoculated with C. gloeosporioides were 60, 41, and 74% less than means for controls, respectively. Equivalent values from inoculations of Type B were less than those of controls by 16, 2, and 26%, respectively (Table 3).

Table 3. Least square means (LSMEANS) for Dry Weight, Weight Change, and Fresh Weight, of Salsola tragus Type A and Type B plants inoculated with Colletotrichum gloeosporioides (Colletotrichum)

Analysis of disease rating data supports the relative impact of C. gloeosporioides on Type A, compared to Type B. Wilcoxon scores were significantly higher for treatment of Type A with C. gloeosporioides than for treatment of Type B (Table 4). Symptoms were noted on individual plants of Type B, but almost all of them (93.3%) were rated either ‘0’ or ‘1’. In contrast, 72% of the individual plants of Type A were rated either ‘3’ or ‘4’.

Table 4. Number of Salsola tragus Type A and Type B plants rated in each disease category at the termination of the studya

4. Discussion

Based on results of this study, genetic background appears to be a significant factor to the potential success of C. gloeosporioides as a biological control agent of Russian thistle. S. tragus Type A was clearly more susceptible than Type B in this study, based on biomass reductions (FWT, DWT, and WTCH; Table 3) and damage (disease ratings at the termination of each study, Table 4). The data also suggested that there is some measurable and statistically significant effect from inoculation of Type B in terms of biomass, but there is practically no visible impact or damage to these plants.

Information from this study underscores the importance of understanding the biology and taxonomy of the target weed. The amount of genetic variation within Russian thistle populations in North America is only beginning to be understood (Ryan and Ayres, 2000). Neither, the number of introductions of Salsola species into North America nor the areas of Europe and Asia from which these introductions originated are known. Formation of novel hybrids in North America by introgression may be a possibility and must be considered as well (Rilke, 1999). In the light of these factors, it is not surprising that biological control agents might be effective against one “type” or species of plant and less effective against others. Current work at the morphological level (F. Hrusa, California Department of Food and Agriculture Sacramento, CA; personal communication) indicates that S. tragus Type B may be a distinct species and supports earlier findings with molecular makers (Ryan and Ayres, 2000). Continuing research suggests the existence of several other variants within S. tragus in California (P. Akers and F. Ryan, respectively, California Department of Food and Agriculture, Sacramento, CA, and USDA-ARS, Fresno, CA; personal communication). This has significant implications for research on biological control of this pest. Further, research on pathogens of Type A is warranted, since, survey data indicates that it is the most common of the known variants. The need for biological control agents specific to Type B, and possibly other variants, has been identified, and it is now possible to screen for agents against Type B.

Differences in susceptibility of rush skeletonweed (Chondrilla juncea L.) also were identified during evaluations of the rust fungus, Puccinia chondrillina Bubak and Syd., for biological control. At least one of two forms in the United States and two of three forms in Australia were resistant to infection by isolates of P. chondrillina that were evaluated (Cullen, 1985; Emge et al., 1981). Despite this, the rust was released in each country with measurable success ( Cullen, 1985; Emge et al., 1981; Supkoff et al., 1988). Since then, either from plant resistance to the rust and other agents or for environmental reasons, skeletonweed has persisted and continues to be a pest targeted for biological control. The situation with S. tragus may result in similar lack of ultimate control due to the filling of ecological niches with a type not susceptible to agents currently under consideration. Until taxonomy and genetics of the target are fully understood in terms of damage from pathogens and insects, there will be little likelihood for success in finding suitable biological control agents.

Optimal conditions for infection of S. tragus by C. gloeosporioides are similar to those reported by others in evaluations of Colletotrichum species for biological control of weeds. Wymore et al. (1988), in determining optimal conditions for inoculation of velvetleaf (Abutilon theophrasti Medik.) with Colletotrichum coccodes (Wallr.) S.J. Hughes, found that the highest disease levels occurred between 18 and 24 h dew at 24 or 30 °C. Likewise, studies with C. gloeosporioides, the agent registered as the mycoherbicide Collego, indicated the best infection of northern jointvetch (Aeschynomene virginica (L. and C.) B.S.P.) occurred between 18 and 32 °C with at least 12 h dew. In subsequent studies, plants inoculated with C. gloeosporioides were given 12 h dew at 28 °C (TeBeest et al., 1978). Dew conditions selected for inoculations in the present study, 16 h at 25 °C, caused the greatest infection and damage to inoculated plants under conditions that did not affect uninoculated controls (Table 2, Fig. 1).

Current research centers on completing the risk assessment of C. gloeosporioides. Development of the rust fungus, U. salsolae, also is of interest, considering that very good infections were noted on Type A in past experiments (Bruckart et al., 2000; Hasan et al., 2001). Additional isolates of U. salsolae are being sought, because of the encouraging preliminary results on Type A.

Acknowledgements The authors acknowledge and thank D.K. Berner for advice on statistical analysis of data and for reviews of drafts by D.K. Berner, D.G. Luster, and Mike Pitcairn.

References

Bruckart, W.L., Luster, D.G., Ryan, F., Schwarczinger, I., Vajna, L. and Sobhian, R., 2000. Getting a grip on the target: experiences with Salsola diseases. In: Spencer, N.R., Editor, , 2000. Proceedings of the Tenth International Symposium on Biological Control of Weeds, Montana State University, Bozeman, MT, p. 675.

Burdon, J.J., Groves, R.H. and Cullen, J.M., 1981. The impact of biological control on the distribution and abundance of Chondrilla juncea in south-eastern Australia. J. Appl. Ecol. 18, pp. 957–966. Full Text via CrossRef

Crompton, C.W. and Bassett, I.J., 1985. The biology of Canadian weeds. 65. Salsola pestifer A. Nels. Can. J. Plant Sci. 65, pp. 379–388.

Cullen, J.M., 1985. Bringing the cost benefit analysis of biological control of Chondrilla juncea up to date. In: Delfosse, E.S., Editor, , 1985. Proceedings of the Sixth International Symposium on the Biological Control of Weeds, Agriculture Canada, Ottawa, pp. 145–152.

Emge, R.G., Melching, J.S. and Kingsolver, C.H., 1981. Epidemiology of Puccinia chondrillina, a rust pathogen for the biological control of rush skeleton weed in the United States. Phytopathology 71, pp. 839–843.

Goeden, R.D. and Pemberton, R.W., 1995. Russian thistle. In: Nechols, J.R., Andres, L.A., Beardsley, J.W., Goeden, R.D. and Jackson, C.G., Editors, 1995. Biological Control in the Western United States, University of California, Berkeley, California, pp. 276–280. Hasan, S., 1985. Search in Greece and Turkey for Puccinia chondrillina strains suitable to Australian forms of skeleton weed. In: Delfosse, E.S., Editor, , 1985. Proceedings of the Sixth International Symposium on the Biological Control of Weeds, Agriculture Canada, Ottawa, pp. 625–632.

Hasan, S., Sobhian, R. and Herard, F., 2001. Biology, impact and preliminary host- specificity testing of the rust fungus, Uromyces salsolae, a potential biological control agent for Salsola kali in the USA. Biocontrol Sci. Technol. 11, pp. 677–689. View Record in Scopus | Cited By in Scopus

Mosyakin, S., 1996. A taxonomic synopsis of the genus Salsola (Chenopodiaceae) in North America. Ann. Missouri Bot. Gard. 83, pp. 387–395. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus

Müller, H., Nuessly, G.S. and Goeden, R.D., 1990. Natural enemies and host-plant asynchrony contributing to the failure of the introduced moth, Coleophora parthenica Meyerick (Lepidoptera: Coleophoridae), to control Russian thistle. Agric. Ecosyst. 32, pp. 133–142. SummaryPlus | Full Text + Links | PDF (593 K) | View Record in Scopus | Cited By in Scopus

Rilke, S., 1999. Revision der Sektion Salsola s.l. der Gattung Salsola (Chenopodiaceae). Bibliotheca botanica, Heft 149. E. Schweizerbart’sche Verlagsbuchhandlung (Nagele u. Obermiller), Stuttgart. p. 189

Ryan, F. and Ayres, D., 2000. Molecular markers indicate two cryptic, genetically divergent populations of Russian thistle (Salsola tragus) in California. Can. J. Bot. 78, pp. 59–67. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus

Saari, L.L., Cotterman, J.C., Smith, W.F. and Primiani, M.M., 1992. Sulfonylurea herbicide resistance in common chickweed, perennial ryegrass, and Russian thistle. Pest. Biochem. Physiol. 42, pp. 110–118. SummaryPlus | Full Text + Links | PDF (756 K) | View Record in Scopus | Cited By in Scopus Schwarczinger, I., Vajna, L. and Bruckart, W.L., 1998. First report of Colletotrichum gloeosporioides on Russian thistle. Plant Dis. 82, p. 1405.

Sobhian, R. and Andres, L.A., 1978. The response of skeletonweed to Cystiphora schmidti and the gall mite Aceria chondrillae. Environ. Entomol. 7, pp. 506–508.

Sobhian, R., Ryan, F.J., Khamaraev, A., Pitcairn, M.J. and Bell, D.E., 2003. DNA phenotyping to find a natural enemy in Uzbekistan for California biotypes of Salsola tragus L. Biol. Control 28, pp. 222–228. SummaryPlus | Full Text + Links | PDF (252 K) | View Record in Scopus | Cited By in Scopus

Supkoff, D.M., Joley, D.B. and Marois, J.J., 1988. Effect of introduced biological control organisms on the density of Chondrilla juncea in California. J. Appl. Ecol. 25, pp. 1089– 1095. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus

TeBeest, D.O., Templeton, G.E. and Smith Jr, R.J., 1978. Temperature and moisture requirements for development of anthracnose on northern jointvetch. Phytopathology 68, pp. 389–393.

Wilken, D.H., 1993. Chenopodiaceae. In: Hickman, J.C., Editor, , 1993. The Jepson Manual: Higher Plants of California, University of California Press, Berkeley, CA, pp. 500–515.

Wymore, L.A., Poirier, C., Watson, A.K. and Gotlieb, A.R., 1988. Colletotrichum coccodes, a potential bioherbicide for control of velvetleaf (Abutilon theophrasti). Plant Dis. 72, pp. 534–537. Full Text via CrossRef

Young, F., Veseth, R., Thill, D., Schillinger, W., Ball, D., 1995. Managing Russian thistle under conservation tillage in crop-fallow rotations. In: Pacific Northwest Conservation Tillage Handbook. Pacific Northwest Extension Publications, PNW492, Washington State University, Pullman. p.12 Young, J.A. and Evans, R.A., 1985. Russian thistle—the weed that won the west. Weeds Today 16 1, pp. 4–7.

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