Life-cycle of Puccinia abrupta var. partheniicola, a potential biological control agent of Parthenium hysterophorus

[ 1°5 ] Trans. Br. mycol. Soc. 88 (1) 105-111 (1987)

Printed in Great Britain

LIFE-CYCLE OF PUCCINIA ABRUPTA VAR. PARTHENIICOLA, A POTENTIAL BIOLOGICAL CONTROL AGENT OF PARTHENIUM HYSTEROPHORUS By H. C. EVANS Commonwealth Institute of Biological Control, Silwood Park, Ascot, Berks SLS 7PY Puccinia abrupta var. partheniicola was found to produce only two spore stages (uredinia and telia) on the composite weed, Parthenium hysterophorus, within its native habitat in north-east Mexico. Conventional methods to induce teliospore germination were unsuccessful. A method is described in which teliospores were subjected to prolonged oxidation by hydrogen peroxide and sodium hypochlorite. The former chemical, in particular, both stimulated teliospore germination and reduced contamination by hyperparasitic micro-organisms, after washing the telia in a concentrated solution (30% wjv) for 30-60 min. By inoculating young plants under greenhouse conditions with basidiospores, pycnia and aecia were obtained. A brief description of these previously unrecorded spore stages is given. The rust is confirmed as being a macrocyclic, autoecious species. Parthenium hysterophorus L. (Heliantheae, Compositae) or false ragweed is an increasingly serious weed of both urban and agricultural land in Australia and India (Towers et al., 1977). Its aggressive weedy habit is causing considerable concern in central Queensland, where extensive areas of rangeland have been invaded in recent years (Haseler, 1976; Dale, 1981). In addition, P. hysterophorus is toxic to animals and hence not acceptable as alternative forage, and is reported to cause allergic responses, such as respiratory malfunction and dermatitis, in susceptible humans (Subba Rao et al., 1978; McClay, 1983). Conventional methods of weed control are uneconomic and only partially effective in the Queensland situation and biological control is considered to be the best long-term solution (Haseler, 1976). Several insects with biocontrol potential have been imported recently into Australia (McClay, 1983) from one of the purported centres of origin of the plant in north-east Mexico (Dale, 1981) and, in order to supplement the effects of these phytophagous insects on P. hysterophorus, the use offungal pathogens is also being evaluated. Disease surveys were undertaken in Mexico in March-May 1983 and November 1984 (Evans, 1983, 1984), during which the rust Puccinia abrupta Diet. & Holw. var. partheniicola (Jackson) Parmelee (Parmelee, 1967) was regularly collected. In some semi-arid, high-altitude localities (1400-1600 m a.s.l.), the fungus was observed to reduce both the vegetative growth of young plants and the seed production of older plants. In these

habitats the rust was found to produce both uredinia and telia in abundance on the leaves, stems and inflorescences. However, in the more humid, lowland and coastal situations, infection was generally light and only scattered uredinia occurred, typically on the older rosette leaves. In none of the collecting sites in Mexico was there evidence to suggest that pycnia and aecia are formed on P. hysterophorus under field conditions, and there has been no report of these stages in collections of the rust from other countries (Parmelee, pers. comm.). Before a biocontrol organism can be considered for importation into Australia, its biology and life-cycle must be fully documented. For P. abrupta var. partheniicola, therefore, it was necessary to obtain evidence to support the hypothesis that the rust is full-cycled and autoecious (Parmelee, 1967; pers. comm.), in order to satisfy the basic quarantine requirements. Since the field surveys had failed to achieve this end, it was concluded that manipulation of the teliospores was the only feasible way of establishing the nature of the life-cycle. The results from the first Mexican survey were not encouraging: none of the germination methods was successful at the uncontrolled room temperatures employed (20-35°C); and there was no evidence of infection when telia were suspended over host plants and subjected to a prolonged wetting and drying regime (Evans, 1983). This paper reports on the experimental procedures used to try to break the dormancy of teliospores of P. abrupta var. partheniicola and thence to elucidate the life-cycle.

Life-cycle of Puccinia abrupta v. partheniicola

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Table 1. Effect of various sterilants on contamination of telia of Puccinia abrupta var. partheniicola and germination of teliospores Immersion time (min) Sterilants Mercuric chloride (0 ' 1 %) Silver nitrate (0' 1 % ) Sodium hypochlorite

5 - (l )*t - (1)

15

30

60

- (1) - (0)

- (0) - (0)

- (0) - (0)

- (3) - (3) + +(1 )

- (3) - (2) + + + + (0)

- (3) - (1) + + + +(0)

1% 10- 1

4%

Hydrogen peroxide 5% 15 % 30 %

- (4) - (3) + (2)

* Estimated germination based on the scale: - not detectable; + germination detected « 1 %) ; + + low ( > 1 %), inconsistent; + + + low (> 1 %), consistent; + + + + moderate ( > 5 %), consistent. t Figures in parentheses indicate degree of contamination: o none or negligible; 1 low; 2 medium; 3 high; 4 limiting, no recording of germination possible several days after incubation due to overgrowth of saprophytes.

MATERIAL AND METHODS

Preliminary germination tests Rust material, collected from Saltillo (Coahuila State, ca 1650 m a.s.l. ) during the first survey in May 1983, was used in the preliminary tests conducted in the U.K. Teliospores, treated as described by Groth & Mogen (1978), which basically involved prolonged washing in water to remove any inhibitors or contaminant organisms, were spread on to tap-water agar (T WA) and incubated at 25, 20,15,12 and 5°. Germination was extremely poor ( > 0'01 %) and occurred only on plates maintained at 15 and 12°. Subsequent tests gave inconsistent and predominantly negative results. Alternative methods were sought, therefore, in order to obtain a reliable method of producing sufficient quantities of basidiospores to carry out inoculation experiments. These included: (a) cycling teliospores through alternate periods offreeze-thaw and wet-dry (Inman, 1971; Gold & Statler, 1983); (b) incorporating autoclaved stems and leaves of P. hysterophorus into TWA seeded with teliospores (K lisiewicz, 1972 );

(c) prolonged cold treatments (Anikster, 19 83; Schubiger et al., 1984); (d) heat treatments (G old & Mendgen, 1983). All the methods proved to be impractical since, during the initial observation period (5 d ), no germination was detected and material held after this period became heavily contaminated and overgrown by bacteria, yeasts and dematiaceous fungi, particularly Alternaria alternata (F r .) Keissler and Cladosporium spp ., despite the presence of chloramphenicol, streptomycin sulphate and pimaricin in the TWA. Benomyl (1000 p.p.m.) also failed to suppress the contaminants. Pre-treatment with a range of standard laboratory sterilants was investigated, which led subsequently to the development of a simple and effective methodology to break teliospore dormancy .

Inducem ent of teliospore germination The experiments were carried out in January and February 1985 using rust-infected plants collected during November 1984 from Ciudad de Maiz (San Luis Potosi State, 1400 m a.s.I.). Pieces of stem

Figs 1-6. Teliospore germination in P . abrupta var. partheniicola following treatment with concentrated hydrogen peroxide (x 1000) . Fig. 1 . Swollen, bleached teliospore, exhibiting first signs of germination, 6 d after treatment . Figs 2-3 . Initiation of germ-tube formation. Fig. 4. Teliospore with two-celled germination and basidiospore. Fig. 5. Germinating basidiospore. Fig. 6. Detail of an early stage in germination. In the upper cell the wall has been breached and the cytoplasm has migrated into the developing germ-tube. In the lower cell the cytoplasm is granular and activated, as evidenced by Brownian movement, and there is a thinning of the inner wall and rupturing ofthe outer wall.

H. C. Evans

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3

6

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Life-cycle of Puccinia abrupta v. partheniicola

and leaf, bearing telia and ca 5-8 mm in length, were placed in McCartney bottles (2 em diam) containing the sterilant and a magnetic flea ( 1 . 5 ern in length) and agitated using a magnetic stirrer (Rodwell Monotherm) at medium speed for 5, 15, 30 or 60 min . The pieces were then transferred with flamed forceps to bottles containing sterile distilled water (SD W) and agitated for 15 min . This was repeated with four changes of SDW. Each sterilant was tested initially at the standard recommended concentration, but this was increased with those chemicals which failed to control contamination (see Table 1) . In the case of silver nitrate, a 0 '1 % solution of sodium chloride was used to remove the sterilant from the tissues. The resultant precipitate of silver chloride was removed by vigorous washing. Tclia were transferred finally to TWA plates, incubated at 12-15° in total darkness for 30 d, and examined daily with a stereoscopic microscope.

micro-organisms. The standard concentration of sodium hypochlorite failed both to suppress contamination and to promote germination. However, prolonged washing in a concentrated solution (10- 14 % active chlorine) reduced the level of contamination and induced germination . Similarly, at low concentrations of hydrogen peroxide, contamination was high and there was no detectable germination. At high concentrations (15- 30 % w/v), contamination was not a problem and there was consistent evidence of teliospore germination. The experiment was repeated on two separate occasions and comparable results were obtained. Germination was slow in all the treatments, and the first evidence of germ-tube formation occurred 3-4 d after incubation (F igs 1-6). The germination rate increased steadily over a period of 9-10 d, but a high proportion of the teliospores remained dormant throughout the observation period.

Inoculation with basidiospore s

Inoculation

Seeds of P. hysterophorus from Australia and Mexico were sown in 5 em diam pots using a mixture of sand, gravel and potting compost and maintained in an isolation plant propagator (Burkhard Manufacturing Co. Ltd; Jenkyn et al., 1973 ) at 25° inside a greenhouse fitted with sodium lamps (Hilux) on a 12 h cycle. In order to comply with U.K. quarantine requirements, the propagator was specially modified so that the airflow was reversed, creating a system in which air was constantly pulled into the individual propagator hoods, over the plants, through ventilation pipes in the pots and finally out via double bacterial filters . The system ensured that any spores introduced into the pots, or subsequently developing within them, did not escape into the atmosphere . Plants were inoculated at 6-8 wk of age, using basidiospore-bearing teliospores which were selected with the aid of a stereoscopic microscope and transferred with a fine camelhair brush to young rosette leaves. Immediately after inoculation, plants were placed in a plastic-lined chamber fitted with a humidifier (Defensor) and maintained at 15-20° and 100 % humidity in total darkness for 24 h . The plants were returned to the propagator and examined daily over a 6 wk period.

Inoculated plants exhibited a range of symptoms : (a) circular, raised, chlorotic areas (6-10 mm diam ) appearing on both leaf surfaces, 11-12 dafter inoculation, later developing circular aggregations of pycnia; (b) discrete pycnial groups, scattered randomly along the leaf margins with little or no chlorosis or other tissue abnormalities; (c) pycnia appearing 23-30 d after inoculation on leaf midribs and petioles, associated with gross tissue distortion and hypertrophy . Characteristic yellow to orange, cup-shaped aecia developed irregularly on or around some of the lesions, 7-20 dafter pycnial appearance.

RESULTS

Germination

The results are summarized in Table 1 . No germination was recorded in treatments with mercuric chloride or silver nitrate, although both chemicals completely eliminated contaminant

Taxonomy

A taxonomic supplement to the description of P. abrupta var. partheniicola (Parmelee, 1967 ) is included here . The rust is macrocyclic and autoecious, all spore stages being produced on P. hysterophorus. Pycnia amphigenous, scattered or grouped in

raised, chlorotic spots, subepidermal, ampulliform to subglobose, yellow becoming brownish-orange with age, 90-120 x 100-140 jlm (F igs 7-8). Aecia amphigenous or caulicolous, in necrotic areas , when densely grouped causing gross hypertrophy and distortion, pulverulent, cupulate, 500-600 Jim diarn ; peridium white , coarsely lacerate, ruptured epidermis conspicuous (F ig. 9). Aeciospores yellow to orange in mass, becoming white when dried, globose to ovoid, (25- )28- 33(- 36) x (20-) 23- 28(- 30 ) tan, occasionally apiculate ; wall

H. C. Evans

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Figs 7-10. Sexual stages of P. abrupta var. partheniicola. Fig. 7. Pycnia forming in raised, chlorotic spots on both sides of the leaf of P. hysterophorus, 18 dafter inoculation with basidiospores (x 130). Fig. 8. Detail of pycnium, showing abundant spermatia and subepidermal origin ( x 205). Fig. 9. T.S. of aecium; note the conspicuous ruptured epidermis forming the wall of the cupulate structure, the peridium has collapsed (x 130). Fig. 10. Aeciospores with verrucae, particularly prominent on lateral walls ( x i ooo),

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Life-cycle of Puccinia abrupta v. partheniicola

yellow, 1'8-3'2/Lm thick, including verrucae, warts irregular in size and shape, typically smaller in lower and upper third and coarse in centre, flat-topped (Fig. 10). Peridial cells white, imbricate, coarsely and densely verrucose, polygonal to irregular, (33-)38-52(-56) x (21-)24-33(-38) pm. DISCUSSION

The teliospores of P. abrupta var. partheniicola, although produced in abundance on P. hysterophorus in the upland, semi-arid valleys of north-east Mexico, seem to be non-functional in these indigenous habitats. The rust apparently cycles through repeated generations of urediniospores. During the field surveys, senescent, telial-bearing inflorescences of P. hysterophorus were commonly found along the edges of ploughed fields and it was assumed that this was the method of rust survival during the extremely dry intercrop period. However, emergent, newly infected plants struggling to establish in the adjacent cultivated land showed no evidence of pycnia or aecia and only uredinia were observed. Microscopic examination of the old telia did not reveal any signs of previous teliospore germination. This apparent redundancy of the teliospore stage and the loss of the sexual stages from the life-cycle within the endemic range of P. abrupta var. partheniicola is analogous to that of P. chondrillina Bubak & Syd., a rust which has been used successfully in Australia for control of the skeleton weed, Chondrilla juncea L. (Cullen et al., 1973; Hasan, 1974). Throughout the area of co-evolution of the weed and the rust in Mediterranean Europe, P. chondrillina produces only uredinia and telia; neither pycnia nor aecia have been reported (Hasan & Wapshere, 1973). However, these workers did manage to germinate the teliospores, produced on rare occasions under conditions of extreme cold, but never elicited infection. Consequently, the rust was imported into Australia with an incomplete or unproven knowledge of its life-cycle, the assumption that P. chondrillina was a full-cycled, autoecious species being based entirely on circumstantial evidence from a few North American records (Arthur, 1929). Ironically, when the rust was introduced recently into the western part of the U.S.A., the sexual stages were found consistently in the field and were readily reproducible under greenhouse conditions (Adams & Line, 1984). During the present study, the life-cycle of P. abrupta var. partheniicola was determined only after an intensive laboratory investigation, which revealed that drastic treatment with concentrated hydrogen peroxide, and to a lesser extent with sodium hypochlorite, is necessary to break teliospore

dormancy. These bleaching agents were also effective in reducing contamination, particularly by saprophytic fungi, which was a limiting factor in the early germination experiments, despite the fact that spores from old telia and those parasitized by Sphaerellopsis filum (Biv-Bern.: Fr.) Sutton were not used. Evidence from tissue sectioning showed that in certain cases Cladosporium mycelium was intimately colonizing the telia and surrounding necrotic tissues, and the fungus appeared to be functioning as a specialized hyperparasite. Removal of the contaminating organisms by standard laboratory sterilants, or by vigorous and sustained washing, failed to promote germination of the teliospores, and thus both hydrogen peroxide and sodium hypochlorite must have been providing an additional stimulatory factor. It is hypothesized that the inducement of germination results from a chemical bleaching or oxidation of the dark teliospore wall, possibly simulating the high uv radiation encountered in the semi-arid, upland regions of Mexico. In such habitats the teliospores may be vestigial, dating from a period when agriculture was not practised and hence the occurrence of susceptible hosts was much more irregular and sporadic, P. hysterophorus being best suited to disturbed environments. Survival for long periods, in the form of thickwalled teliospores, and the staggered germination of these resting spores, following prolonged exposure to sunlight, would have been evolutionarily advantageous under such conditions. This study formed part of a project (PAR.475) funded by the Queensland Department of Lands (Land Administration Commission). Permission to publish the paper was given by the Director, Alan Fletcher Research Station, Sherwood, Queensland. The U.K. research was carried out under MAFF licence number PHF 873/36. REFERENCES

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pathogens and their hosts. Annals of Applied Biology 73,9- t3 · KLiSIEWICZ, J . M. ( 1972). Effect of host plant materials and temperature on germination of teliospores of Puccinia carthami, Phytopathology 62, 436--438. MCCLAY, A. S. (1983). Natural enemies of Parthenium hysterophorus L. (Cornpositae) in Mexico : Final Report 1978-t983. C.I.B .C . Unpublished Report, 1-50. PARMELEE, J. A. (1967). The autoecious species of Puccinia on Heliantheae in North America. Canadian Journal of Botany 45, 2267-2328. SCHUBIGER, F . X., DEFAGO, G., SEDLAR, L. & KERN, H . (1984). Host range of the haplontic phase of Uromyces rumicis . Proceedings of the Sixth International Symposium on Biological Control of Weeds , Vancouver, Canada, 1-17 . SUBBA RAo, P . V., MANGALA, A., TOWERS, G. H. N . & RODRIGUEZ, E . (1978). Immunological activity of parthenin and its disastereoisomer in persons sensitized by Parthenium hysterophorus L. Contact Dermatitis 4, 199-203. TOWERS, G . H . N., MITCHELL, T. C., RODRIGUEZ, E ., BENNETT, F. D. & SUBBA RAo, P. V. (1977). Biology and chemistry of Parthenium hysterophorus L., a problem weed in India. Journal of Scientific and Industrial Research 36, 672-684.

(R eceived for publication 17 June 1986 )