Root Infection and Systemic Colonization of Maize by Colletotrichum graminicola

Wild-type strain M1.001-BH (also known as CgM2) (13) and its derivative transgenic strains were used in all the experiments. Cultures were maintained at 25°C on potato dextrose agar medium (PDA; Difco Laboratories, Detroit, MI) with continuous illumination from a fluorescent light source. Long-term storage at −80°C involved suspending conidia in 7% skim milk mixed with silica gel (unless specified otherwise, all reagents were obtained from Sigma, St. Louis, MO) (57).

Several GFP-tagged strains of C. graminicola were obtained by transforming fungal protoplasts with plasmid pGFP (21, 35), which contains the GFP gene driven by the promoter from the Aspergillus nidulans gdp gene as well as the hph gene, which confers resistance to hygromycin B (35). Protoplast preparation and transformation were performed as described by Thon et al. (56), except that 100 mg of lysing enzymes per ml of 0.7 M NaCl was used to digest conidial cell walls. Protoplasts were released after 4 to 5 hours of digestion. Selection for transformants was performed using PDA amended with 100 mg/liter hygromycin B (PDA+hyg).

Transformants were genetically purified by preparing a dilute suspension of conidia in sterile H₂O and spreading the suspension on PDA. After incubating for 24 h, a single colony was selected. This process was repeated once. The transformants were assayed for colony morphology, growth rate, and virulence. Transformants were assayed for GFP activity by fluorescence microscopy using techniques and equipment described below. Overall colony morphology and color were compared to the wild-type strain, while growth rate was measured on PDA in 150-mm petri dishes by measuring colony radius daily for seven consecutive days after inoculation. Foliar virulence was assayed using the method described by Thon et al. (56), and root virulence was assayed using the methods described below. The transformants were also tested for mitotic stability by subculturing for several weeks on PDA in the absence of hygromycin and then transferring them to PDA+hyg and assaying them for GFP activity.

Maize inbred lines B73 (susceptible to ALB [13]) and H99 (highly resistant to ALB [62]) were inoculated with C. graminicola, and infection was followed over a time course. To study systemic colonization, we infected maize lines B73, H99, and Mo940 (also susceptible to leaf blight [13, 61, 62]). Prior to being planted, all seeds were treated by coating them with the fungicide trifloxystrobin (Flint 50; Bayer CropScience) and allowing them to dry at room temperature for 24 h. The seeds were washed in 70% ethanol for 5 min and immediately rinsed with water to remove the fungicide before being inoculated. We noted no adverse effect on the development of C. graminicola after this treatment (data not shown). Three root inoculation techniques were employed. For studies of early root colonization, we inoculated maize roots from seeds that had been germinated in petri dishes. Seeds were placed in petri dishes in sets of four on wet, autoclaved filter paper and watered every 2 days with a solution of 0.5 g liter⁻¹ Peter's fertilizer (Peter's 20-20-20; AG Spectrum, IA). Seedlings were inoculated 3 days postgermination using either a root soak method or an agar plug method. For the root soak procedure, conidia were collected from 14- to 21-day-old cultures of M1.001-BH-gfp or M1.001-BH. The conidia were filtered through three layers of cheesecloth and then washed with two rounds of resuspension in sterile distilled water (dH₂O) followed by centrifugation (3,000 rpm, 3 min, room temperature). The roots were soaked for 1 hour in a suspension of 10⁷ conidia ml⁻¹. For the agar plug inoculation method, cultures of M1.001-BH-gfp were grown on PDA for 14 days. Agar plugs (approximately 10 mm in diameter, cut from the leading edge of a 14-day-old colony) were placed directly on exposed seedling roots. Once inoculated, the seedlings were observed daily under a microscope starting 1 day postinoculation (dpi).

The late stages of root colonization, including the colonization of upper plant parts, were studied by inoculating plants with the protocol described by Dufresne and Osbourn (10). Briefly, five agar plugs from 14-day-old M1.001-BH-gfp cultures were placed in polycarbonate 50-ml centrifuge tubes containing 35 ml of autoclaved vermiculite clay. Another 5 ml of vermiculite was placed over the agar plugs, one maize seed was added, and an additional 5 ml of vermiculite was added to create approximately 1 cm of vermiculite cover. Ten seedlings were prepared for each trial. For a control, one set of five seedlings was prepared for each trial in tubes inoculated with sterilized PDA plugs. Each tube was watered with sterile dH₂O and sealed with Parafilm. Once the seedlings germinated, the Parafilm was removed, and the seedlings were watered every other day using 0.5 g liter⁻¹ Peter's fertilizer (20-20-20). The experiment was performed four times.

For experiments requiring plants more than 3 weeks old, seedlings were first grown in vermiculite and inoculated with agar plugs as described above. The plants were transferred to 3- or 10-liter pots containing a mixture of 40% peat, 40% loam, 10% vermiculite, and 10% compost. The greenhouse temperature was 25 to 27°C, and daylight was supplemented with light from fluorescent tubes to provide 14 h of continuous light. The light intensities in the greenhouse during the day were between 120 and 200 microeinsteins m⁻². The plants used in greenhouse experiments were irrigated daily with water and twice a week with the Peter's fertilizer solution.

Tissue samples were dissected from inoculated and mock-inoculated plants at 42 dpi. Two- to 3-mm-diameter sections were cut from leaves (first to third leaf), stems (first and second aboveground internodes), and roots (mesocotyl, radicle, and nodal root) (Fig. 1). The dissected tissues were surface sterilized for 45 seconds in 10% sodium hypochlorite and rinsed three times in sterile dH₂O. Each dissected tissue section was divided into 10 pieces and placed on one-half-strength PDA containing 1.5 ml liter⁻¹ of 80% lactic acid, 100 μg ampicillin/ml, and 100 μg hygromycin B/ml. The samples were incubated at 25°C for 10 days and examined daily by bright-field and fluorescence microscopy. This experiment was replicated three times, with each replicate consisting of seven inoculated plants and seven mock-inoculated plants. From each plant, 10 samples from each tissue were cultured and examined for the presence of C. graminicola. The tissue was considered infected if C. graminicola was found in any of the 10 samples.

The images shown in Fig. 2 to 5 (with exceptions noted below) were acquired with an Olympus BX60 microscope outfitted with 10×, 40×, and 100× objectives (Olympus America Inc., Melville, NY). Observation of GFP fluorescence employed an Endow GFP long-pass filter with excitation and emission wavelengths of 470 and 500 nm, respectively, and a dichroic mirror at 495 nm (model no. 41018; Chroma Technology, Rockingham, VT). Images were captured with a Q-FIRE digital camera (Q-Imaging, Burnaby, BC, Canada) using Q-Capture software version 2.56. Alternatively, the image shown in Fig. 3C was acquired using an Olympus BX51 microscope outfitted with a DP70 camera (Olympus America Inc., Melville, NY). A detailed description of the features of this system has been described earlier (54). The images in Fig. 4E to G and that in Fig. 5J were acquired with an Olympus SZX9 stereomicroscope outfitted with an Olympus DP70 camera. Adobe Photoshop CS2 version 9.02 (Adobe, Mountain View, CA) was used to prepare images for publication.

Between 1 and 28 dpi, infected roots were monitored for fungal colonization. For each time point examined, at least 20 inoculated and 5 mock-inoculated roots were assayed from each of four independent experiments. The tissue samples were placed on a microscope slide, submerged in a water droplet, covered with a glass coverslip, and visualized as described above.

Tissue samples for Fig. 5G were prepared using previously described methods (37). Briefly, the tissues were fixed in Methacarn (55) for 24 h at 4°C, dehydrated in a graded ethanol series, and embedded in paraffin. Five-micrometer sections were cut with a microtome, stained with lactophenol aniline blue (0.01% [wt/vol]), and examined with bright-field microscopy.

MS production was induced in vitro using the protocol described by Sanogo and Pennypacker (51). Briefly, agar plugs from 7- to 10-day-old cultures were transferred to petri dishes containing PDA. The petri dishes were sealed with Parafilm and incubated in darkness at 28°C for 6 weeks. The MS were collected with a sterile toothpick and cleaned of medium, mycelia, and spores by rolling them on celite medium (3% agar and 1% diatomaceous earth as a mild abrasive) (26). Following the celite treatment, the MS were surface sterilized by agitating them gently for 45 seconds in 10% sodium hypochlorite. The MS were collected in a Büchner funnel fitted with filter paper and washed three times by alternately filling the funnel with 100 ml sterile dH₂O and applying a vacuum. Individual cleaned MS were used to inoculate 3-day-old maize seeding roots. Seedlings were watered every 2 days with 0.5 g liter⁻¹ Peter's 20-20-20 fertilizer. The root tissue was observed microscopically between 2 and 10 days postinfection.

We used 6 μg of plasmid DNA to transform 10⁷ protoplasts and obtained 38 hygromycin-resistant transformants. Thirty-five of the transformants exhibited GFP expression, though variation in expression levels was detected among the transformants. Ten transformants with high levels of fluorescence were selected for further analysis and were purified by single-spore isolation. Microscopic analysis confirmed that in all of these strains, high levels of fluorescence were observed in the mycelium, falcate conidia, and oval conidia (data not shown). Transformants showed phenotypes indistinguishable from that of the wild type with respect to colony morphology, pigmentation, spore production, and growth rate (data not shown). Foliar pathogenicity assays performed with these transformants resulted in lesion development that was indistinguishable from that of the wild-type strain, and transformants were stable in media with or without hygromycin B (data not shown). A single strain was randomly selected for use in further experiments.

Assays of infected plants showed that GFP fluorescence could be detected in infected plants at least 18 weeks after infection. GFP fluorescence was observed for all nonmelanized fungal structures during maize leaf and root colonization, consistent with observations of axenic cultures.

To determine whether C. graminicola could infect maize roots, we performed infection assays followed by a time course of microscopic observations. The infection assays were performed with the ALB-susceptible maize line B73 as well as the ALB-resistant line H99. Throughout the time course study, no differences in infection patterns between leaf blight-resistant and -susceptible lines were observed.

Early infection and colonization events were observed by means of germinating maize seeds in petri dishes and inoculating the 3-day-old seedlings with colonized agar plugs. As soon as 1 day postinfection, hyphae colonized the radicle surface (Fig. 1 and 2A and B). At 2 dpi, hyphae grew along the epidermis parallel to the longitudinal axis of the root (Fig. 2C). At this point, lateral, swellings could be seen at the junction between plant cells (Fig. 2C and D). Shortly thereafter, the lateral swellings became melanized, and hyphae could be observed within the subtending epidermal cells (Fig. 2D). We refer to these swellings as hyphopodia, following the definition provided by Howard (22), which describes appressoria as structures that develop from swellings at the tips of conidial germ tubes and hyphopodia as structures that arise from mature vegetative hyphae. No appressoria, structures typical of C. graminicola foliar infections, were observed on the root surface, even when spore suspensions were used as the inoculum. Instead, conidia germinated to form hyphae (Fig. 2E).

At 3 dpi, the hyphae became thick and melanized, resembling microhyphae or runner hyphae (20), and could be observed extensively colonizing the root surfaces (Fig. 2F). At this stage, fluorescence was generally not observed because the accumulation of melanin blocked the light, although less mature fluorescent hyphae could be found in association with the melanized hyphae. The hyphae extensively colonized the root surface, forming a network around the root (Fig. 2G). At 3 dpi, hyphae were also observed invading the intercellular spaces of epidermal roots cells (Fig. 2H), although no visible disease symptoms could be found on the roots. Once inside the root, the hyphae swelled and spread intracellularly into the cytosol of the cell through specific points of contact with other epidermal and cortical cells. Hyphae became greatly constricted as they passed through the cell wall (Fig. 2I). Hyphae could also be observed growing intracellularly among the epidermal and cortical cells (Fig. 2J). Infected epidermal and cortical cells frequently became packed with hyphae (Fig. 2J to L). The hyphae proliferated within the cell, filling most of the intracellular space (Fig. 2J), and eventually became melanized (Fig. 2K). The hyphae appeared to be confined to the infected cell until the intracellular space was completely filled with hyphae (Fig. 2J and K). Only certain epidermal and cortical cells appeared to become colonized in this manner, while the surrounding cells remained uninfected, resulting in a mosaic pattern of plant cell colonization (Fig. 2L). This pattern of plant cell colonization continued for 4 to 25 dpi.

Many root pathogens are reported to preferentially infect their hosts through specific regions of the roots (18, 31, 42). For example, in the Fusarium verticillioides-maize root interaction, the main sites of penetration are the lateral roots (42). Specificity for certain regions of the roots may indicate that there is tissue-specific variation in factors that determine susceptibility to C. graminicola and thus may be helpful in identifying those factors. To determine if C. graminicola also has site of attachment or penetration preference, roots from 3-day-old plants were dipped in a spore suspension, transferred to a petri dish, and observed by fluorescence microscopy for 24 to 72 h. Hyphae could be seen colonizing the surfaces of mature roots, root caps, root elongation zones, and root hairs (data not shown), indicating that there was no root infection site preference.

The formation of acervuli began with the development of stromata within the epidermal cells. At 4 to 6 dpi, stromatic tissue formed inside epidermal cells, giving rise to conidiophores and setae (Fig. 3A and B). These structures subsequently expanded, resulting in mechanical rupture of the cuticle and the formation of masses of falcate conidia and characteristic setae. MS tended to form near acervuli (Fig. 3B). Interestingly, falcate conidia were formed in acervuli as soon as 5 dpi on the root surfaces but were also found filling epidermal cells and root hairs at 10 dpi (Fig. 3C to F). Cells that were filled with oval conidia could also be found (Fig. 3G). The conidia were viable and often they could be observed germinating within the epidermal cells (Fig. 3H) and the root hairs (not shown). Brown discoloration was evident on roots during the advanced stages of colonization (approximately 6 dpi and later). Microscopic examination of the roots indicated that the brown discoloration was due to the presence of plant cells that were densely packed with melanized hyphae and acervuli.

At 21 dpi, hyphae continued to spread intracellularly through specific points of contact with other cells (data not shown). At this time point, extensive root areas were covered with acervuli and setae (Fig. 3I) and large root areas were discolored. Despite the extensive colonization by melanized hyphae and the presence of acervuli, necrotic lesions were not observed on the roots at this time point. Microscopic observation of plants at 28 dpi revealed that the entire root system (Fig. 1), including the radicle, mesocotyl, and nodal, lateral, and seminal roots of both B73 and H99 were infected. Even root sections that showed no visible symptoms of colonization, such as brown discoloration, were colonized by the fungus (Fig. 3J). The red pigmentation of the root is characteristic of the maize line B73. After 42 dpi, some signs of root necrosis could be observed.

Sections from infected and uninfected roots of both leaf blight-susceptible and -resistant maize lines were examined at various time points to determine the rates and extents of colonization. At 7 dpi in both maize lines, the pathogen colonized the epidermis and the cortical cells, both intercellularly and intracellularly (Fig. 3K and L). At 14 dpi, the whole root cortex was heavily colonized, until the fungus reached the endodermis (Fig. 3L and M). The endodermis appeared to delay the progress of the hyphae, and it was not until 21 dpi that the fungus was observed colonizing the vascular cylinder (Fig. 3N and O), where it could be observed colonizing the endodermis, sieve cells (phloem) and xylem (Fig. 3N). While xylem vessels were colonized by hyphae (Fig. 3O), no blockage of the vascular system was observed, nor did the plants exhibit wilting symptoms, typically found with vascular diseases. Some yellow-green fluorescence was observed in the piths of the infected roots; however, the emission spectrum of this fluorescence was different from the GFP emission spectrum, indicating that the cells accumulated autofluorescing material, similar to what has been observed for the Fusarium verticillioides-maize root interaction (42). In the uninfected roots, yellow autofluorescence was observed in the maize epidermis, endodermis, phloem, and xylem (Fig. 3P).

As soon as 5 dpi, we observed MS attached to hyphae on root surfaces (Fig. 4A to C). Size and shape varied, but MS continued to increase in number and size over time (Fig. 4A to C and data not shown). The MS were generally small (5 to 20 μm) and appeared to be comprised of aggregated, compact, melanized hyphae. At 20 dpi, a large number of them could be observed on the root (Fig. 4D).

We investigated whether the MS were viable sources of inoculum. Because of their small size, isolation of MS from infected plant tissue was not feasible, so an in vitro MS induction system described previously for other Colletotrichum spp. was employed. The MS produced in vitro were numerous and had morphology similar to that found for infected plant tissue (Fig. 4E and F). Masses of salmon-orange conidia inside mucilage could be observed on the surfaces of the MS (Fig. 4F). The MS produced in culture were surface cleaned and sterilized (see Materials and Methods) to remove hyphal fragments and conidia from the surface before applying them to uninfected roots. The MS produced in culture were able to infect plant roots. Myceliogenic (Fig. 4G) and sporogenic (not shown) germinations of the MS were observed, and runner hyphae and hyphopodia could be observed colonizing the root surface (Fig. 4G).

To determine whether C. graminicola could colonize aboveground plant parts upon the infection of roots, we attempted to isolate the fungus from infected plants. Plants were infected with C. graminicola strain M1.001-BH-gfp. At 42 dpi, segments of above- and belowground plant parts were dissected, surface sterilized, and cultured on acidified PDA+hyg. The cultures exhibiting C. graminicola colony morphology were examined by fluorescence microscopy to confirm their identities. The number of successful C. graminicola isolations for each tissue type is shown in Table 1. Colletotrichum graminicola was not recovered from any part of the mock-inoculated plants but could be recovered from 97.8% of the roots and from 28.3% of the aboveground parts of root-inoculated plants. The fungus was recovered more than twice as often from the aerial parts of leaf blight-susceptible lines B73 (40.0%) and Mo940 (33.3%) as from the leaf blight-resistant line H99 (15.8%).

Examination of the aerial plant parts at 28 dpi under fluorescence microscopy revealed that the adventitious roots, senescent coleoptiles, and senescent first and second leaf sheaths were frequently colonized (Fig. 1 and 5 A to C). These results were consistently observed among all of the maize lines tested (B73, H99, and Mo940). Senescent coleoptiles were heavily colonized (Fig. 5B and C). Acervuli and conidia were also found on the aerial plant organs. Within the green, living leaf sheath tissue, hyphae were also occasionally observed inside plant cells (Fig. 5D) and leaf trichomes (Fig. 5E).

Often, the limiting step in the movement of plant pathogenic fungi from infected roots to the upper parts of the plant is the transition of the fungus from the seedling crown to the stalk, and the transition usually takes place through the vascular tissue (40, 42). Therefore, transverse sections of leaves and stems were used to determine whether C. graminicola colonized vascular tissue, parenchymatous tissue, or both tissue types. While the strong autofluorescence of the plant tissue made direct observation of C. graminicola in these sections difficult, culturing the stem transverse sections on isolation medium revealed the presence of hygromycin-resistant GFP-expressing C. graminicola from individual vascular bundles (Fig. 5F). To determine whether the fungus was localized within specific tissues within the leaves of infected plants, some samples were fixed, embedded, and stained with aniline blue and visualized with bright-field microscopy. Hyphae appeared to be restricted to the phloem tissue of the vascular bundle of the leaf (Fig. 5G). We also observed the production of lobed hyphopodia in the infected, senescent leaf sheaths (Fig. 5H to J). These structures originated from melanized hyphae and are attached to the plant leaves. Younger hyphopodia were fluorescent, but over time, melanin accumulation masked the fluorescence (Fig. 5H and I). At 6 to 8 weeks postinfection, necrotic, water-soaked lesions began to appear on the roots of infected plants, but no symptoms were observed on upper plant parts under the tested conditions (data not shown).