Mycol. Res. 100 (2): 165-172 (1996) Printed in Great Britain 165

Infection process of Colletotrichum gloeosporioides f. sp. malvae on Malvaceae weeds

LOUISE MORIN1+, JO-ANNE L. DERBY' AND ERIC G . KOKKO' Agriculture and Agri-Food Canada, Research Station, P.O. Box 440, Regina, Saskatchewan, Canada, S4P 3A2 'Agriculture and Agri-Food Canada, Research Station, P.O. Box 3000, Lethbridge, Alberta, Canada, TIJ 4B1

Colletotrichum gloeosporioides f. sp. malvae is highly pathogenic on Malva pusilla and Malva parviflora but not on Malva neglecta and Abutilon theophrasti. Germination of conidia was higher on agar discs than on leaf surfaces of the Malvaceae hosts and safflower. Germination, however, was considerably reduced on agar discs when the number of conidia applied was increased. Timing of the germination process and morphology of pre-penetration structures were similar on the leaf surface of the five host species examined. Slightly more than 4% of melanized appressoria developed infection structures within epidermal cells of M . pusilla, A . theophrasti and safflower. Levels of penetration were lower (< 2%) on M . parvifira and M . neglecta. Colletotrichum gloeosporioides f. sp. malvae penetrated plant cuticles directly and produced infection structures within epidermal cells 31-36 h after inoculation. The mode of infection of C. gloeosporioides f. sp. malvae involved intracellular vesicles, large-diameter primary hyphae that constricted at transcellular penetration sites and secondary hyphae. Development of primary hyphae and production of secondary hyphae was extensive on the susceptible hosts M . pusilla and M . parviflora. In the moderately resistant hosts M . neglecta and A . theophrasti colonization was stopped by a hypersensitive reaction of cells adjacent to initial infection sites and no secondary hyphae were produced. Our study strongly suggests that determinants of host-pathogen compatibility or incompatibility do not operate during the pre-penetration phase of C. gloeosporioides f. sp. malvae, but are activated a few days after successhl penetration.

In 1992, Collefofrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae was registered in Canada under the bioherbicide trade name BioMalm for use on M a l v a pwilla Sm. (round- leaved mallow) (Makowski & Mortensen, 1992). This fungus was first isolated in 1982 from diseased M. pusilla growing in a glasshouse (Mortensen, 1988). The disease was later found on this weed in several locations in Saskatchewan and Manitoba, Canada. Under natural conditions, the disease is endemic and usually develops late in the growing season. The potential to develop C. gloeosporioides f. sp. malvae as a bioherbicide was established in controlled environment, glasshouse and field studies (Makowski, 1987, 1993; Morten- sen, 1988; Makowski & Mortensen, 1989; Mortensen & Makowski, 1989). Its registration was followed by extensive research for the development of a large-scale submerged fermentation system for its production (Cunningham, Kuiack & Komendant, 1990).

In host-specificity tests, C . gloeosporioides f. sp. malvae was found to be highly pathogenic on M a l v a parviflora L. (small- flowered mallow), another Malvaceae weed (Makowski, 1987; Mortensen, 1988). The fungus infected the other closely- related weeds, M a l v a neglecta Wallr. (common mallow) and Abutilon theophrasti Medic. (velvetleaf), to a lesser extent (Makowski, 1987, 1993; Mortensen, 1988). It produced only

* Present address: Manaaki Whenua - Landcare Research, Private Bag 921 70, Auckland, New Zealand.

small necrotic lesions on other Malvaceae and on crop plants such as safflower (Carthamus finctorius L.; Asteraceae) (Mortensen, 1988; R. M. D. Makowski & K. Mortensen, unpublished data).

Enhancing the pathogenic activity of BioMalB on mod- erately resistant Malvaceae weeds has been contemplated recently, because it would substantially increase the market size for this bioherbicide. For instance, a bioherbicide t o control A . theophrasfi has considerable market potential as this weed is a major problem in corn (Zea mays L.) and soybean (Glycine m a x (L.) Merr.) in the U.S.A. (Spencer, 1984).

Limited knowledge exists on the infection process of C. gloesporioides F. sp. malvae (Makowski, 1987). The infection strategies of plant pathogenic fungi involve several stages such as attachment and germination of propagules, differen- tiation into specialized pre-penetration structures (e.g. appres- soria), penetration of host cells, development of infection hyphae and colonization of plant tissues (Bailey e f a]., 1992). At any of these stages, slight inherent or induced differences in morphology, biochemistry or physiology between plants can have major repercussions on the establishment of a compatible interaction with a pathogen and hence expression of disease symptoms. Resistance mechanisms restricting colonization of moderately resistant hosts by C . gloeosporioides f. sp. malvae may be deployed at any stage of the infection process and act simultaneousIy or sequentially. Detailed knowledge of the infection process of C . gloeosporioides f. sp. malvae may provide

Colletotrichum gloeo~~orioides on Malvaceae weeds 166

helpful information to direct research towards optimizing the effectiveness of BioMalB.

This study was undertaken to obtain a better understanding of the basis of the host-pathogen relationship between C. gloeosporioides f. sp. malvae and highly susceptible or mod- erately resistant Malvaceae hosts. The infection process of the fungus on four Malvaceae weeds was examined using scanning electron and light microscopy. In addition, the pre-penetration and penetration phases of the fungus were assessed quan- titatively. Safflower was included in these experiments as a non-Malvaceae and moderately resistant host. The findings provide clues to explain differences in disease severity between Malvaceae weeds. Possible strategies to enhance efficacy of BioMalB on M. pusilla and closely related weeds are discussed.

MATERIALS AND METHODS

Plant production

Seeds of M. pusilla and A. fheophrasti were collected from plants harvested at Regina (Saskatchewan) and Ste-Anne-de- Bellevue (QuCbec), respectively. Seeds of M. parviflora and M. neglecfa were collected at Tracy, California. Seeds of safflower cv. St08 were obtained from J. W. Bergman (Eastern Agri- cultural Research Centre, Sidney, MT). Malvaceae seeds were scarified by removing a small portion of the seed coat with a scalpel. They were then placed onto moist filter papers contained in 9 cm diam. glass Petri dishes and incubated in the dark at 24& 1 OC for 48 h. Safflower and germinated Malvaceae seeds were sown in a soil-vermiculite-peat moss mixture (I : 1 : 1) in 12.5 cm diam. plastic pots (4-5 seeds per pot) and grown in a controlled environment chamber (model E-15, Conviron, Winnipeg, Canada) with a 16 h photoperiod (335 i.lE m-2 s-') and 24/20$1° day/night temperatures. Plants were fertilized weekly with a nutrient solution (N-P-K, 30-10-10).

Znoculum production and inoculation of plants

Conidia of C. gloeosporioides f. sp. malvae (ATCC 20767, BioMalB isolate) frozen at - 70' in a skim milk and glycerol solution (D. 0 . TeBeest, pers. comm.) were transferred onto Potato Dextrose Agar (PDA) contained in 9 cm diam. Petri dishes and incubated in the dark at 24 1' for 1 wk. SufFicient inoculum for experiments was produced by transferring conidia onto several PDA plates and streaking the surface with an inoculating loop. Plates were incubated as above for 10-15 d. Conidia were harvested by washing the surface of the agar with distilled water. The density of the suspension of conidia was determined using a haemocytometer and adjusted to 2 x lo6 conidia ml-l with distilled water (5 x lo6 or 1 x lo7 conidia ml-' in experiments for scanning electron and light microscopy). The suspension of conidia was sprayed onto plants (2 to 3- or 3 to d e a f stage) until run-off using a spray atomizer (type H-5, Paasche Airbrush Ltd, Chicago, IL) at a constant air pressure of 200 kPa. Inoculated plants were placed in a dark dew chamber (model E-54U-DL, Percival, Boone, IA) set at 20* lo for 24 h (22 h in experiment on

penetration of leaves) and then transferred to a controlled environment chamber (conditions as above).

Germination of conidia on agar discs

Droplets (10.2 pl) of various suspensions of conidia (1 x lo5, 5 x lo5, 1 x lo6, 5 x lo6, 1 x lo7, 5 x 10' conidia ml-l) in distilled water were placed on 1.5 % water-agar discs (12 mm diam.), allowed to air dry for 5-10 min and covered with coverslips. The glass slides supporting the agar discs were placed in 9 cm diam. glass Petri dishes in an incubator (model 125L, Conviron, Winnipeg, Canada) set at 24 f with a 12 h dark (beginning of the incubation period)/l2 h light regime (29-49 pE m-2 s-') for 24 h. After incubation, the coverslips were removed and 2.5 p1 droplets of lactophenol-cotton blue were placed on the agar discs to stop further germination. Conidia were considered to have germinated when the length of the germ-tube was greater than the width of the conidium, or when a sessile appressorium was produced. Several randomly selected fields of view were examined using a compound microscope (Leitz Diaplan, model GMBH, Wild Leitz, Wetzlar, Germany) until a total of 200 conidia per replicate had been assessed.

Germination of conidia on plants

Water-agar discs (12 mm diam.) were sprayed with the suspension of conidia used on plants and placed in glass Petri dishes in the dew chamber. At 24 h after inoculation of plants, 12 mm diam. discs were cut from the middle of the second oldest leaf of the four Malvaceae and safflower and placed (adaxial surface uppermost) onto moist filter papers in glass Petri dishes until assessment. Leaf discs were transferred onto glass slides and a droplet of Auramine-0 fluorescent stain (3 g Auramine-0 [ICN Biochemicals, Cleveland, OH], 70 ml glycerol, 30 g phenol, 930 ml distilled water) was pIaced onto their surface (McRae, 1989). Stained leaf discs were im- mediately examined using a compound microscope equipped with epifluorescence (exciter filter BP420-490, dichromatic mirror RKP 510, suppression filter LP 520; Fluorescence Power Pack, model 990002, Ludi Electronics Products Ltd, Scarsdale, NY) because the stain is not specific for fungi and components of the leaf soon fluoresce. This staining method has the advantage of not disturbing too many conidia on the leaf surface. Agar discs were treated with lactophenol-cotton blue as described above. Two hundred conidia per leaf and agar discs were examined from several randomly selected - fields of view. Germination of conidia was assessed as described in the previous section.

Scanning electron microscopy

At 24, 48 and 72 h after inoculation, leaf pieces (approx. 0.5 cm2) were sampled from the four Malvaceae weeds and immediately fixed by immersion in 1 % glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.0), overnight at 4'. Samples were brought to room temperature (21 + lo) in the morning for 1 h. Alternatively, leaf pieces were fixed for

Louise Morin, Jo-Anne L. Derby and Eric G. Kokko 167

10-15 min at room temperature in a heptane/glutaraldehyde mixture prepared as follows: a I: 1 mixture of 4% aqueous glutaraldehyde was shaken in 0.05 M sodium phosphate buffer (pH 7-0) and heptane for 1 min; the mixture was left to stand until the two phases separated; the top heptane phase, which had absorbed some of the fixative, was decanted and used. Leaf pieces were then removed from the heptanelglutar- aldehyde mixture and immersed in 4% glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.0) for 2-4 h at room temperature. After both fixation methods, the specimens were washed in 0.05 M sodium phosphate buffer (pH 7.0; three rinses of 10 min each) at room temperature, dehydrated in a graded ethanol series, critical-point dried with liquid CO, as the transitional fluid (Polaron E3100), mounted on SEM stubs in colloidal silver cement and sputter-coated (Denton Vacuum Desk-1) with gold. The specimens were examined and photographed using a Hitachi S-570 scanning electron microscope.

Light microscopy

Leaf pieces (0.5-1.0 cm2) were sampled from the four Malvaceae weeds every hour during the first 12 h after inoculation, stained with droplets of Auramine-0 fluorescent stain and examined using a compound microscope equipped with epifluorescence (as described above). Some pieces were directly mounted in water and examined using a compound microscope to detect melanized appressoria. At 22, 24, 31, 36, 46, 48, 60, 64, 70, 72, 108, 112 and 136 h after inoculation, samples of leaf pieces were cleared and stained in a solution containing Chlorazol Black E (Sigma Chemical Company), ethanol, chloroform, lactic acid, phenol and chloral hydrate for 16-24 h (Keane, Limongiello & Warren, 1988). The leaf pieces were then rinsed in distilled water and kept in a saturated solution of chloral hydrate for 6 (experiment on penetration of leaves) or 18 h. The vials were left on the bench at room temperature during the whole process. The whole-leaf pieces were subsequently mounted in 50% glycerol and examined using a compound microscope.

Penetration of leaves

At 48 h after inoculation, three leaf discs (12 rnm diam.) were cut from the second oldest leaf of the four Malvaceae weeds and safflower, cleared and stained as described above, mounted in 50% glycerol and examined using a compound microscope. For each leaf disc, up to a maximum of 200 appressoria were assessed for penetration by recording the presence of an infection structure within the epidermal cell below the appressorium. Data collected on the three leaf discs sampled from one plant were pooled for statistical analysis.

Analysis of data

A completely randomized design with four replicates per treatment was used in all experiments. Each experiment was performed twice. When a relationship was observed between the variances and the means of each treatment, percentage data were subjected to an arcsin transformation before an

analysis of variance (Steel & Torrie, 1980). In the experiment evaluating the effect of inoculum density on germination of conidia on agar discs, curvilinear functions were fitted to the data by generating polynomial regression equations using the actual data (no transformation was required). Results from the trials of each experiment were pooled when homogeneity of variances was detected using Barlett's test (Steel & Torrie, 1980) and when no significant interaction between trials and treatments was observed. Significant differences between treatment means were established with Tukey's HSD test (cc = 0.01).

RESULTS

Germination of conidia on agar discs

The ability of conidia to germinate decreased significantly (P < 0.01) as the inoculum density increased (Fig. I). A density of 1 x lo5 conidia ml-' achieved the maximum level of germination (95-99%) observed. The effect of crowding on germination of conidia was more severe in trial 1 (Fig. I). Several melanized appressoria and some penetration pegs were produced.

Pre-penetration phase on plants

The germination process and pre-penetration structures of C. gloeosporioides f. sp. malvae were similar on the leaf surface of all Malvaceae weeds (Figs 2-5). Conidia began to germinate 3-4 h after inoculation by producing a single germ-tube (1-2 ym diam.) (Figs 2,3), which always emerged at a distance from the region of the former point of attachment of the conidiurn with the conidiophore. At 5-6 h after inoculation, germ-tubes had differentiated at their tip into simple, globose to spherical appressoria (3.5-47 x 4.3-5.7 m), which had not yet melanized (Figs 2, 3). Appressoria were predominantly sessile (Fig. 3). A septum developed between the appressorium

5 5.5 6 6.5 7 7.5 8 Inoculum density (Log conidia ml-I)

Fig. 1. Effect of inoculurn density on germination of conidia OF Collefotrichurn gloeosporioides f. sp. malvae on water-agar discs. Trials were not combined because a significant (P < 0.01) interaction between trials and treatments was observed. Data points represent means of four replicates. Regression equations are y = 817.001- 213.336x+13.993x2 for trial 1 (-) and y = 302.159-38.539~ for trial 2 (----) where y = percentage of germination and x = log of inoculurn density.

Colleto~richtlm gloeosporioides on Malvaceae weeds 168

Figs 2-5. Scanning electron micrographs of the infection process of Coiletotrichum gloeosporioides f. sp. malvae on leaves of Malvaceae weeds. Fig. 2. Germinated conidium (C) with a germ-tube (GT) that has differentiated into an appressorium (A) on Malva pwilla at 24 h after inoculation. Germ-tubes were never seen to emerge from the vestigial point of attachment (P) of the conidium with the conidiophore. Note the septum (S) separating the germ-tube and the appressoriurn. Fig. 3. Germinated conidia on M. pusilla at 24 h after inoculation. The germ-tube of one conidium appears to penetrate the host through a stomatal pore (ST). Note the extracellular matrix (M) around the sessile appressoria of the other conidia. Fig. 4. Appressorium on Abutilon theophrasti at 24 h after inoculation. Extracellular matrix around the base of the appressorium is easily detectable. Fig. 5. Germinated conidium with a sessile appressoriurn on Malva parviflora at 72 h after inoculation. Note the collapsing epidermal cell (E) underneath the appressorium.

and the conidium or germ-tube (Fig. 2). A t 10-12 h after observed (Fig. 3). An extracellular matrix was present around inoculation, appressoria had become brownish, which indicated appressoria at the site of attachment on the leaf surface (Figs that melanization had occurred. Appressoria were produced 3, 4). anywhere o n the epidermis, irrespective of the surface N o statistically significant (P < 0.01) difference in ger- topography. Penetration through stomatal pores was rarely mination was detected between conidia placed onto the leaf

Louise Morin, Jo-Anne L. Derby and Eric G. Kokko 169

Table 1. Germination of conidia of Colletotrichum gloeosporioides f. sp. malvae on the leaf surface of Malvaceae weeds and safflower*

Germination of Plant species conidiat (%)

Control (agar discs) 53.70 a* Malva parviflora 15.06 b Abutilon theophrasti 13.69 b Malva pusilla 13.00 b Malva neglects 11.06 b Carthamus tinctorius 9.38 b

* Results are from pooled experiments. t Conidia were considered to have germinated when the length of the

germ-tube was greater than the width of the conidium or when a sessile appressorium was produced. * Treatments associated with the same letter have means that are not significantly ( P < 0.01) different, according to Tukey's HSD test.

surface of the Malvaceae weeds and safflower (Table I). Germination of conidia on leaves, however, was 7 1 4 3 % less than on water-agar discs. Most germinated conidia produced appressoria on leaf surfaces.

Penetration of plants and development of infection sites

The first signs of penetration on Malvaceae hosts were observed at 31-36 h after inoculation (Fig. 6). Penetration occurred directly through the cuticle and epidermal cell wall. Penetration pegs could not be detected with the whole-leaf clearing and staining technique. No subcuticular fungal colonization was seen. Sometimes globose intracellular infection vesicles, slightly smaller in diameter than appressoria, were present within epidermal cells (Fig. 6), but most infection vesicles were irregularly shaped (Fig. 7). One or several intracellular primary hyphae (4.5-7.0 pm diam.) began to develop from the vesicle at 46-48 h after inoculation (Fig. 7). By 72 h after inoculation, the large-diameter intracellular primary hypha had expanded by penetrating adjacent epidermal (Fig. 8) and mesophyll (Fig. 9) cells. Primary hyphae were noticeably constricted at the site of penetration between cells (Figs 8, 9, 10). Initial development of infection structures was similar on all Malvaceae plants examined. Hypersensitive death of the first epidermal cell penetrated by the fungus was not observed on any of the hosts.

Initial macroscopic symptoms expressed as small necrotic flecks developed on M. pusilla and M. parviflora at 84-96 h after inoculation. By 108 h after inoculation, necrotic lesions expanded and were surrounded by a chlorotic halo. In contrast, only a few small necrotic flecks ever developed on leaves of A. theophrasti and M. neglecta. However, severe stem lesions sometimes occurred on A. theophrasti.

Intracellular primary hyphae with constricted transcellular penetration sites were still present at 136 h after inoculation at the margin of spreading infection sites on M. pwilla (Fig. 10). As early as 72 h after inoculation, thin secondary hyphae (1-5-3.0 pm diam.) were developing from normal or heavily septate primary hyphae (Fig. 11) at the foci of infection sites on M. pusilla and M. parviflora. These secondary hyphae appeared to grow intra- and intercellularly and were not constricted as they passed from cell to cell. The collapsed epidermal cell shown in Fig. 5 probably indicates that

secondary hyphae have started to develop in the focus of this putative infection site on M. parviflora. Extensive development of primary hyphae and production of secondary hyphae were not seen on M. neglecta or A. fheophrasti. In contrast, dead epidermal cells adjacent to successfully penetrated cells (commonly observed on these hosts at 108-112 h after inoculation) appeared to restrict further development of primary hyphae (Fig. 12).

A low percentage (< 5 %) of melanized appressoria of C. gloeosporioides f. sp. malvae developed infection structures within epidermal cells of the various hosts tested (Table 2). Penetration was significantly ( P < 0.01) higher on safflower and A. fheophrasti than on M. parviflora and M. neglecfa. Percentage penetration on M. pusilla was not significantly (P < 0.01) different from that recorded on the other plants examined.

DISCUSSION

The present study compared the infection process of C. gloeosporioides f. sp. malvae on highly susceptible (M. pttsilla, M. parviflora) and moderately resistant (M. neglecta, A. theo- phrasti) hosts. This particular pathogen has received only limited attention at the microscopic level (Makowski, 1987), although the infection process of C. gloeosporioides has been investigated in great detail on various hosts (Brown, 1977; TeBeest, Templeton & Smith, 1978; Chau & Alvarez, 1983; Daykin & Milholland, 1984; Ogle, Gowanlock & Irwin, 1990; Coates, Muirhead, Irwin & Gowanlock, 1993).

Conidia of C. gloeosporioides f. sp. malvae germinated rapidly on both agar discs and leaf surfaces. Germination, however, was considerably reduced on agar discs as the number of conidia increased. The inhibitory effect of crowded environ- ments on germination has been reported for Collefotrichum spp. and has been linked to the presence of the extracellular matrix, which is produced in the acervulus during conidio- genesis (Mondal & Parberry, 1992). Self-inhibitors of ger- mination have been isolated from the matrix of Collefotrichum spp. (Leite & Nicholson, 1993). The inhibition of germination of conidia on plant surfaces by self-inhibitors present in the matrix when large numbers of conidia are present should be investigated in bioherbicide research since high inoculum densities are generally sprayed onto target weeds.

Colletotrichum gloeosporioides f. sp. malvae germinated and produced appressoria equally well on the leaf surface of all hosts examined. Germination, however, was much lower on leaves than on agar discs. The presence of fungitoxic leachates of plant origin (Tukey, 1970) may be responsible for this reduced rate of germination on leaves. The unusual high pH in the phylloplane of Malvaceae plants may also have inhibited germination (Harr, Guggenheim & Boller, 1984). Moreover, antagonistic micro-organisms affecting germi- nation on the leaf surface could be involved. Micro-organisms isolated from the leaf phylloplane or fruit surface of various plants have been bound to inhibit germination of Collefotrichum spp. by competition for nutrients and space or by antibiosis (Lenne & Brown, 1991; Koomen & Jeffries, 1993).

The morphology and abundance of appressoria of C. gloeosporioides f. sp. malvae were similar on the different

Collefotrichttm gloeosporioides on Malvaceae weeds 170

Figs 6-11. Light micrographs of the infection process of Colletotrichum gloeosporioides f. sp. malvae on leaves of Malva pwilla Fig. 6. Globose intracellular infection vesicle (V) within an epidermal cell at 36 h after inoculation. Note the appressorium and conidium on the surface of the epidermis. Fig. 7. Irregularly shaped intracellular infection vesicle with three primary hyphae (PH) developing at 48 h after inoculation. Fig. 8. Growth of an intracellular primary hypha into a neighbouring epidermal cell at 72 h after inoculation. Note the transcellular penetration site (arrow). Fig. 9. Intracellular primary hypha invading a mesophyll cell at 72 h after inoculation. Fig. 10. Intracellular primary hypha in several epidermal cells at the margin of an infection site at 136 h after inoculation. Note transcellular penetration sites (arrows). Fig. 11. Heavily septate primary hyphae in the focus of an infection site at 136 h after inoculation. Note initial development of secondary hyphae (SH). Fig. 12. Light micrograph of an infection site of Colletotrichum gloeosporioides f. sp. malvae on a leaf of Malva neglecta at 108 h after inoculation. Note the hypersensitive reaction (HR) of the epidermal cell adjacent to the cell infected with an intracellular primary hypha.

Louise Morin, Jo-Anne L. Derby and Eric G. Kokko 171

Table 2. Penetration from melanized appressoria of Colletotrichum gloeosporioides f. sp. malvae on leaves of Malvaceae weeds and safflower*

Penetrationt (%)

Safflower 4.62 a* Abutilon theophrasti 4.31 a Malua pusilla 4.01 ab Malva parviflora 1.60 b Malua neglecta 1.32 b

Results are from pooled experiments. t The presence of an infection structure within the epidermal cell below

a melanized appressorium was considered as successful penetration. $ Treatments associated with the same letter have means that are not

significantly (P < 0.01) different, according to Tukey's HSD test.

Malvaceae weeds studied. As observed in several other species of Colletotrichum (Chau & Alvarez, 1983; O'Connell & Bailey, 1991; Van Dyke & Mims, 1991; Coates et al., 1993), appressoria were generally surrounded by an extracellular mucilage. This slimy matrix is believed to play an important role in adhesion and protection of appressoria (Bailey et al., 1992). Melanization of appressoria of C. gloeosporioides f. sp. rnalvae, which is likely to be essential for successful penetration directly through the epidermis (Kubo & Furusawa, 1991), took place on all plants examined regardless of the host species. This strongly suggests that determinants of host-pathogen compatibility or incompatibility do not operate during the pre-penetration phase of C. gloeosporioides f. sp. malvae. Similar results were reported for C. gloeosporioides on Styiosanfhes guianensis (Aubl.) Sw. and various cultivars of Sfylosanthes scabra Vog. (Vinijsanun, Irwin & Cameron, 1987; Trevorrow, Irwin & Cameron, 1988). This contrasts with results from other studies where germination and appressorium formation were reduced or completely inhibited on resistant hosts (Lenni & Brown, 1991).

Colletotrichum gloeosporioides f. sp. malvae penetrated plant cuticles directly and produced infection structures within epidermal cells. The subcuticular hyphae seen in C. gloeo- sporioides infecting other plants (Brown, 1977; Chau & Alvarez, 1983; Daykin & Milholland, 1984; Vinijsanun et al., 1987; Ogle et al., 1990) were not observed in this study. The colonization process of C. gloeosporioides f. sp. malvae on susceptible host tissue, which involved intracellular infection vesicles, large diameter primary hyphae constricted at transcellular penetration sites and secondary hyphae resembled the mode of infection of Collefotrichurn lindemuthianum (Sacc. & Magn.) Briosi & Cav. on French bean (Phaseolus vulgaris L.) (O'Connell, Bailey & Richmond, 1985; O'Connell & Bailey, 1991). Ultrastructural studies have revealed the hemibiotrophic nature of C. lindemuthianum infections where 'the sequence of transient biotrophic phase followed by slow senescence and eventual death of the infected cells is repeated' (O'Connell & Bailey, 1991; Bailey et al., 1992). Although our study may suggest that biotrophy occurs at the early stages of infection by C. gloeosporioides f. sp. rnalvae, extensive electron mi- croscopy studies are necessary to obtain direct evidence of its putative hemibiotrophic status.

Low levels of penetration (< 5 %) from melanized appres- soria were recorded on all the hosts inoculated with C. gloeosporioides f. sp. malvae. Similar low penetration levels have

been reported for C. gloeosporioides by TeBeest ef al. (1978), Vinijsanun ef al. (1987) and Ogle et al. (1990). More refined microscopy studies are required to reveal the factors preventing infection pegs from penetrating epidermal cells.

No consistent difference between levels of penetration for susceptible and moderately resistant hosts was observed in our study. Our findings differ from those of other studies on C. gloeosporioides, where significantly higher levels of pen- etration were recorded on susceptible hosts (Irwin, Trevorrow & Cameron, 1984; Vinijsanun et al., 1987; Trevorrow ef al., 1988). Nevertheless, after the successful penetration of an epidermal cell in moderately resistant hosts, infection hyphae of C. gloeosporioides f. sp. malvae progressed much slower than in highly susceptible Malvaceae weeds. Moreover, extensive colonization was eventually stopped by what appeared to be a hypersensitive reaction of cells contiguous to those infected by the fungus. Indeed, recognition of incompatible interactions between C. gloeosporioides f. sp. malvae and moderately resistant hosts seemed to take place a few days after successful penetration of one or a few epidermal cells. The relatively large limited necrotic lesions produced by C. lindernuthianum on moderately resistant bean cultivars seem to be related to the duration of the biotrophic phase during infection (O'Connell & Bailey, 1988). A similar phenomenon may regulate symptom development in Malvaceae.

Efficiency of plant defence mechanisms against diseases can be altered by herbicides (Altman, Neate & Rovira, 1990; Livesque & Rahe, 1992) and growth regulators (Nickerson, Tworkoski & Luster, 1993). Johal & Rahe (1990) found that glyphosate partly suppressed accumulation of phytoalexins in beans infected by C. lindemuthianum. In addition, the presence of glyphosate enabled infection hyphae to resume growth from cells that had died as a consequence of hypersensitive reaction (Johal & Rahe, 1988). Other work suggested that the growth regulator thidiazuron may impair the peroxidase- mediated defence response of A. fheophrasfi to the pathogen Colletofrichum coccodes (Wallr.) S. Hughes (Nickerson et al., 1993). Since resistance of M. neglecfa and A. fheophrasfi to C. gloeosporioides f. sp. malvae is expressed during the post- penetration phase, it is possible that the efficacy of BioMalm on these weeds could be increased by exploiting interactions between herbicides and the defence system of plants.

The low germination and penetration rates of C. gloeo- sporioides f. sp. malvae recorded on M. pusilla in our study indicated that most conidia sprayed onto plants were ineffective in initiating disease. This explains why a high inoculum dosage (2 x 107 conidia m-2) of this fungus is recommended to achieve adequate development of disease symptoms in the field (Mortensen & Makowski, 1989). Research on ecological behaviour of potential bioherbicides in the phylloplane of target weeds as well as improvement of mass-production systems and formulation is essential to develop efficient and economically acceptable bioherbicides. Products containing highly stable and pathogenic propagules, formulated in a way that enhances germination, appressoria formation and penetration, could lead to a reduction in the dosage of inoculum required to cause severe disease on target weeds and consequently a reduction in production cost of bioherbicides (Auld & Morin, 1995).

Colleto~richctm gloeosporioides on Malvaceae weeds 172

We acknowledge Mr Byron Lee and Ms Fran Leggett for their technical assistance in preparing specimens and using the scanning electron microscope. We also wish to thank Dr Gordon Thomas and Dr Allan Smith (Agriculture and Agri- Food Canada) and Ms Joanna Orwin (Landcare Research) for reviewing the manuscript. The senior author was the recipient of a Natural Sciences and Engineering Research Council of Canada (NSERC) fellowship in a Canadian Government laboratory during the period of these studies.

R E F E R E N C E S

pathogens, with special reference to glyphosate. Annual Review of Phyt~~athology 30, 57%02.

Makowski, R. M. D. (1987). The evaluation of Malva pusilla Sm. as a weed and its pathogen Colletotrichum gloeosporioides (Penz.) Sacc. f. sp. malvae as a bioherbicide. Ph.D. Thesis, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Makowski, R. M. D. (1993). Effect of inoculum concentration, temperature, dew period. and plant growth stage on disease of round-leaved mallow and velvetleaf by Colletotrichum gloeosporioides f. sp. malvae. Phytopathology 83, 1229-1234.

Makowski, R. M. D. & Mortensen, K. (1989). Colletotrichum gloeosporioides f. sp. malvae as a bioherbicide for round-leaved mallow (Malva pusilla): conditions for successful control in the field. In Proceedings of the 7th International Symposium on Biological Control of Weeds (1988) (ed. E. S. Delfosse), pp. 513-522. Rome, Italy.

Altman, J.. Neate. S. & Rovira, A. D. (1990). Herbicide-pathogen interactions ~ a k ~ ~ ~ k ~ , R. M. D. & M ~ ~ ~ ~ ~ ~ ~ ~ , K. (1992). ~h~ first mycoherbicide in and mycoherbicides as alternative strategies for weed control. In Microbes canada: ~ o ~ ~ e ~ o ~ r i c ~ u m g~oeospor;oides f , sp. for round-leaved mallow and Products as Herbicides (ed. R. E. Hoagland), PP. 240-259. control. In Proceedings of the 1st International Weed Control Congress (1992) American Chemical Society: Washington, DC, U.S.A. (compiled by R. G. Richardson), pp. 298-300. Melbourne, Australia.

Auld, B. A. & Morin, L. in the of McRae, C. F. (1989). The feasibility of using Colletotrichum orbiculare as a bioherbicides. Weed Technology (in press). mycoherbicide to control Xanthium spinosum (Bathurst burr). Ph.D. Thesis,

Bailey, I. A.. O'Connell. R. 1.. Pring. R. I. & Nash, C. (1992). Infection ufiversitv of N~~ ~ ~ ~ l ~ ~ d , ~ ~ i d ~ l ~ , NSW, ~ ~ ~ t ~ ~ l i ~ , strategies of Colletotrichum species. In Colletotrichum: Biology, Pathology and Control (ed. J. A. Bailey & M. J. Jeger), pp. 8&120. C.A.B. International: Wallingford, U.K.

Brown, G. E. (1977). Ultrastructure of penetration of ethylene-degreened Robinson tangerines by Colletotrichum gloeosporioides. Phytopathology 67, 315-320.

Chau, K. F. & Alvarez, A. M. (1983). A histological study of anthracnose on Carica papaya. Phytopathology 73, 1113-1116.

Coates, L. M., Muirhead, I. F., Irwin, J. A. G. & Gowanlock, D. H. (1993). Initial infection processes by Colletotrichum gloeosporioides on avocado fruit. Mycological Research 97, 1363-1370.

Cunningham, J. E., Kuiack, C. & Komendant, K. E. (1990). Viability of Penicillium bilaji and Colletotrichum gloeosporioides conidia from liquid cultures. Canadian Journal of Botany 68, 2270-2274.

Daykin, M. E. & Milholland, R. D. (1984). Histopathology of ripe rot caused by Colletotrichum gloeosporioides on muscadine grape. Phytopathology 74, 1339-1341.

Harr, J., Guggenheim, R. & Boller, T. (1984). High pH-values and secretion of ions on leaf surfaces: a characteristic of the phylloplane of Malvaceae. Experientia 40, 935-937.

Irwin, J. A. G., Trevorrow, P. R. & Cameron, D. F. (1984). Histopathology of compatible interactions involving biotypes of Colletotrichum gl~eos~orioides that cause anthracnose of Stylosanthes spp. Australian Journal of Botany 32, 631-640.

Johal, G. S. & Rahe, J. E. (1988). Glyphosate, hypersensitivity and phytoalexin accumulation in the incompatible bean anthracnose host-parasite in- teraction. Physiological and Molecular Plant Pathology 32, 267-281.

Johal, G. S. & Rahe, 1. E. (1990). Role of phytoaiexins in the suppression of resistance of Phaseolw vulgaris to Colletotrichum lindemuthianum by glyphosate. Canadian Journal of Plant Pafhology 12, 225-348.

Keane, P. J., Limongiello, N. & Warren, M. A. (1988). A modified method for clearing and staining leaf-infecting fungi in whole leaves. Ausfralasian Plant Pathology 17, 37-38.

Koomen, I. & Jeffries, P. (1993). Effects of antagonistic microorganisms on the post-harvest development of Colletotrichum gloeosporioides on mango. Plant Paihology 42, 230-237.

Kubo, Y. & Furusawa, 1. (1991). Melanin biosynthesis. Prerequisite for successful invasion of the plant host by appressoria of Colktotrichum and Pyricularia. In The Fungal Spore and Disease Initiation in Plants and Animals (ed. G. T . Cole & H. C. Hoch), pp. 205-218. Plenum Press: New York, U.S.A.

Leite, B. & Nicholson, R. L. (1993). A volatile self-inhibitor from Colletotrichum graminicola. Mycologia 85, 945-951.

Lennk, J. M. & Brown, A. E. (1991). Factors influencing the germination of pathogenic and weakly pathogenic isolates of Colletotrichum gl~eos~orioides on leaf surfaces of Stylosanthesguianensis. Mycological Research 95, 227-232.

LCvesque, C. A. & Rahe, J. E. (1992). Herbicide interactions with fungal root

" . Mondal, A. H. & Parberry, D. G. (1992). The spore matrix and germination

in Colletotrichum mwae. Mycological Research 96, 592-596. Mortensen, K. (1988). The potential of an endemic fungus Colletotrichum

gl~eos~orioides, for biological control of round-leaved mallow (Malva pusilla) and velvetleaf (Abutilon theophrasti). Weed Science 36, 473-478.

Mortensen, K. & Makowski, R. M. D. (1989). Field efficacy at different concentrations of Colletotrich~mgloeos~orioides f. sp. malvae as a bioherbicide for round-leaved mallow (Malva pwilla). In Proceedings of the 7th International Symposium on Biological Control of Weeds (1988) (ed. E. S. Delfosse), pp. 523-530. Rome, Italy.

Nickerson, R. G., Tworkoski, T. J. & Luster, D. G. (1993). Colletotrichum coccodes and thidiazuron alter specific peroxidase activities in velvetleaf (Abutilon theophrasti). Physiological and Molecular Plant Pathology 43, 47-56.

O'Connell, R. J. & Bailey, J. A. (1988). Differences in the extent of fungal development, host cell necrosis and symptom expression during r a c e cultivar interactions between Phaseolus vulgaris and Colletotrichum linde- muthianum. Plant Pathology 37, 351-362.

O'Connell, R. J. & Bailey, J. A. (1991). Hemibiotrophy in Colletotrichum lindemuthianum. In Electron Microscopy of Plant Pathogens (ed. K. Mendgen & D. E. Lesemann), pp. 211-222. Springer-Verlag: Berlin, Germany.

O'Connell, R. J., Bailey, J. A. & Richmond, D. V. (1985). Cytology and physiology of infection of Phaseolw vulgaris by Colletotrichum linde- muthianum. Physiological Plant Pathology 27, 75-98.

Ogle, H. J., Gowanlock, D. H. & Irwin, J. A. G. (1990). Infection of Stylosanthes guianensis and S. scabra by Colletotrichum gloeosporioides. Phytopathology SO, 837-842.

Spencer, N. R. (1984). Velvetleaf, Abutilon theophrasti (Malvaceae), history and economic impact in the United States. Economic Botany 38, 407-416.

Steel, R. G. D. & Torrie, J. H. (1980). Principles and Procedures of Statistics. McGraw-Hill: New York, U.S.A.

TeBeest, D. O., Templeton, G. E. & Smith, R. J. (1978). Histopathology of Colletotrichum gloeosporioides f. sp. aeschynomene on northern jointvetch. Phytopathology 68, 1271-1275.

Trevorrow, P. R., Irwin, J. A. G. & Cameron, D. F. (1988). Histopathology of compatible and incompatible interactions between Colktotrichum gloeo- sporioides and Stylosanthes scabra. Transactions of the British Mycological Society 90, 421-429.

Tukey, H. B. (1970). The leaching of substances from plants. Annual Review of Plant Physiology 21, 305-324.

Van Dyke, C. G. & Mims, C. W. (1991). Ultrastructure of conidia, conidium germination, and appressoriurn development in the plant pathogenic fungus Colletotrichum truncatum. Canadian Iournal of Botany 69, 2455-2467.

Vinijsanun, T., Irwin, J. A. G. & Cameron, D. F. (1987). Host range of three strains of Colletotrichum gloeosporioides from tropical pasture legumes, and comparative histological studies of interactions between type B disease- producing strains and Stylosanthes (non-host) and S. guianensis (host). Australian Journal of Botany 35, 665477.

(Accepted 26 June 1995)