Colonization of maize roots by C. graminicola is similar to that of root pathogens.
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
). 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
Infection of plant roots leads to colonization of aerial plant organs.
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
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
). 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).