This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.
We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.
Spatial and temporal analyses at the cellular level are critical for understanding the physiology and cytology of fungal-plant interactions. Foliar tissues that have been chemically fixed1,2,3or cleared and stained4, as well as artificial membranes5, have been used in the past to investigate the cytology of foliar pathogen development and plant-fungal interactions. However, investigation of infection events in living host tissues in real-time without fixation or clearing is challenging due to technical issues related to the preparation of optically transparent samples for imaging.
A detached leaf sheath inoculation protocol was developed in the late 1940s for bright field microscopic investigation of resistance of living rice epidermal cells to the rice blast fungus Magnaporthe oryza6. More recently, detailed molecular, physiological, and cytological observations of host colonization by Colletotrichum and Magnaporthe species have been greatly facilitated by combining modified versions of this leaf sheath method with fungal transformants expressing fluorescent proteins, and high-performance live-cell imaging protocols, including epifluorescence and confocal microscopy7,8,9,10,11,12,13.
This paper details an optimized inoculation protocol using detached maize leaf sheaths for observation of infection processes by hemibiotrophic and necrotrophic foliar fungal pathogens. We have specifically used it to study Colletotrichum graminicola (C. graminicola), the causal agent of anthracnose leaf blight and stalk rot, and Stenocarpella maydis, which causes Diplodia leaf blight and stalk rot. However, the method should be applicable to other hemibiotrophic and necrotrophic foliar fungal pathogens. Cytological and physiological responses during infection and colonization events in these excised leaf sheaths are similar to those in entire leaf blades12,14,15. Furthermore, hemibiotrophic colonization of sheath epidermal cells by C. graminicola is similar to colonization of stalk pith cells16,17. Detached sheaths show greater synchronicity and experimental reproducibility of fungal penetration and colonization than leaf blades or stalk pith tissues14,16,17,18. Most maize varieties can be used for this protocol. However, inbreds or hybrids with excessive purple pigments in the sheaths are less suitable since the pigments interfere with imaging. Golden Jubilee sweet corn has been particularly useful for our studies because untreated seeds are commercially available, the plants are highly susceptible to many foliar diseases, and they grow well in the greenhouse. The first epidemics of anthracnose stalk rot in the United States resulted in the total loss of sweet corn crops in Indiana in the 1970s19,20. This leaf sheath inoculation method can be applied to directly observe and quantify fungal growth and development in living vs. locally killed plant cells, to demonstrate resistance reactions in compatible/incompatible responses to fungal infection, and to test interactions between fungal strains on the same sheath in real-time.
NOTE: The workflow for the method is shown in Figure 1.
Figure 1: Steps in the optimized inoculation protocol using detached maize leaf sheaths. Spore suspension preparation, leaf sheath inoculation, and sample preparation for live-cell microscopy are highlighted in green (A), purple (B), and orange (C) boxes, respectively. Created with BioRender.com. Please click here to view a larger version of this figure.
1. Plant and fungal material
2. Leaf sheath inoculations
Figure 2: Glass-wool filter unit preparation. (A) A 0.5 cm x 0.5 cm glass-wool ball is placed inside microcentrifuge tube 1 that has its conical bottom removed. (B-C) The filter tube is then placed into microcentrifuge tube 2 to generate an assembled filter unit for spore suspension preparation. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 3: Method of cutting a non-skirted 96-well PCR plate. (A) PCR plate cut into six support racks, 8 x 2 wells. An example of a single sheath support is depicted in (B). Leaf sheaths are laid horizontally on the support. Created with BioRender.com. Please click here to view a larger version of this figure.
Figure 4: Sheath inoculation method. Single drop of inoculum directly applied to the adaxial surface of the sheath section. Please click here to view a larger version of this figure.
Figure 5: Sheath incubation method. Inoculated leaf sheaths placed horizontally in a support rack inside a glass Petri plate containing moistened filter paper. Please click here to view a larger version of this figure.
3. Live-cell microscopy
The examples below describe representative outcomes following the use of the maize leaf sheath inoculation method. These examples demonstrate the ease, speed, and precision with which observation and comparison of maize-fungus interactions can be accomplished in real-time with this optimized assay. Live-cell imaging also allows the extraction of quantitative information, providing a useful tool for comparative molecular, cytological, and physiological studies. Further details may be found in the original publications cited for each successful application.
Example data 1: Use of staining and plasmolysis in inoculated leaf sheaths for evaluation of fungal status and detection of plant responses to fungal infection
Maize leaf sheaths have been used to visualize and compare the infection processes of Colletotrichum graminicola and Stenocarpella maydis in living host cells with bright field microscopy, allowing detailed descriptions of colonization patterns and time courses (unpublished results; Figure 6).
These studies revealed that S. maydis rapidly invades epidermal cells directly via undifferentiated hyphal swellings, unlike C. graminicola which produces melanized appressoria. Once inside the epidermal cells, both pathogens proceed from cell to cell by passing through apparently intact cell walls (Figure 6A,B).
Sheaths can be stained with trypan blue or DAPI to evaluate the nature and extent of colonizing fungal hyphae more easily (Figure 6). Trypan blue staining reveals that C. graminicola initially invades via thick primary hyphae that move between cells through very narrow connections. Later, the fungus switches to a necrotrophic stage, producing thinner secondary hyphae in the center of the colony (Figure 6 C-E)12,14,16,17.
Figure 6: Unstained or stained inoculated maize leaf sheaths imaged with bright field or epifluorescence microscopy. (A) Intracellular hyphae 48 h post-inoculation (hpi) with Stenocarpella maydis. Infection occurs via slightly swollen hyphal tips (arrow). (B) Intracellular hyphae 48 hpi with Colletotrichum graminicola. Infection occurs via melanized appressoria (arrows). (C) Hyphae of C. graminicola, stained with trypan blue, progressing cell-to-cell at the advancing colony edge via narrow connections (arrow), 72 hpi. (D) Closer view of trypan-blue stained hypha of C. graminicola at 72 hpi, passing through the maize cell wall via a narrow connection (arrow). (E) Hyphae of C. graminicola, stained with trypan blue, in the colony center 72 hpi. Narrower secondary hyphae can be seen emerging from the thicker primary hyphae (arrows). (F) Hyphae of C. graminicola 72 hpi, at the advancing colony edge on a leaf sheath stained with DAPI. The stained fungal and plant nuclei fluoresce under UV light. Scale bars in each panel equal to 50 µm. Micrographs obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.
Sheaths can be stained with neutral red and/or subjected to plasmolysis to study the developmental cytology and viability of maize cells during infection and colonization by pathogenic fungi (Figure 7).
Colletotrichum graminicola is a hemibiotrophic fungus that invades living maize cells with thick primary hyphae that are separated from the host cytoplasm by a membrane (Figure 7A-C)12,14,16,17. Stenocarpella maydis, on the other hand, is a necrotrophic fungus that produces a phytotoxin23 and kills the epidermal cells (which become granulated and fail to plasmolyze) before it invades them (Figure 7D).
Maize defense against C. graminicola and other foliar pathogens involves the accumulation of reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2)12,15. To investigate the oxidative chemistry of plant-pathogen interactions, 3,3′-diaminobenzidine (DAB) can be used for cell staining24. DAB is oxidized by H2O2, producing a dark brown precipitate that is visible in the maize leaf sheaths (Figure 7 E,F)12,24. Production of the pigment is an indicator of an oxidative burst related to a host resistance response.
Figure 7: Stained and/or plasmolyzed inoculated maize leaf sheaths imaged with bright field microscopy. (A) Leaf sheath inoculated with C. graminicola 24 hpi and subjected to plasmolysis and staining with neutral red. Living maize cells beneath appressoria (arrows) plasmolyze and stain, demonstrating that the pathogen does not kill the cells prior to invasion. (B) Leaf sheath 48 hpi with C. graminicola, subjected to plasmolysis and staining with neutral red. Maize cells that are colonized by primary hyphae (arrow) are still alive, establishing that C. graminicola invades cells as a biotroph. (C) Leaf sheath 48 hpi with C. graminicola, showing hyphae moving cell-to-cell at the advancing edge of the colony. Sheaths have been plasmolyzed but not stained. Uncolonized cells that are adjacent to the colony edge (arrows) continue to plasmolyze. (D) Leaf sheath 24 hpi with S. maydis, prior to penetration. The cells beneath the germinated spore (arrow) appear granulated and fail to plasmolyze, indicating the S. maydis kills them prior to penetration and that it behaves as a necrotroph. (E) Leaf sheath of resistant maize inbred Mp305 24 hpi with C. graminicola and stained with DAB. Dark staining indicates a strong oxidative response of the single cell in the center of the image, while halos of brown pigment can be seen surrounding most of the individual appressoria, and granulation can be observed in the cells at the top of the photo. (F) A closer view of the halos of brown pigment accumulated around appressoria, and deposition of pigment in the maize cell walls nearby (arrows). Scale bars in each panel equal to 50 µm, except for (F) which is 20 µm. Micrographs obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.
Example data 2: Live-cell imaging with fungi expressing fluorescent proteins
Fungal hyphae expressing cytoplasmic fluorescent proteins can be visualized at high resolution in live sheath cells without the need for any stains by using an epifluorescence or confocal microscope (Figure 8).
Fluorescent proteins can be targeted to various fungal organelles, e.g., the mitochondria, nuclei, or the endomembrane system, in order to track their locations and activities in the living fungal cells in planta (Figure 8C)25,26,27,28 (unpublished results). Fluorescent proteins can also be driven by different fungal promoters to serve as reporters for various functions: for example, RFP driven by the BiP chaperone secretion stress response protein is shown (Figure 8D)29 (unpublished results). The use of leaf sheaths enables the visualization of individually tagged fluorescent fusion proteins that are accumulated and secreted by fungal hyphae as they colonize host cells8,9 (Figure 8E). Transgenic maize lines expressing fluorescent proteins targeted to various cellular compartments, e.g., nuclei or plasma membrane, are also available30,31, and can be used for inoculations to evaluate the effects of infection on maize cellular structures e.g., nuclei. Use of these transgenic maize lines demonstrated that nuclei in uninvaded maize cells typically migrate to the cell wall that is nearest to cells that are already colonized by C. graminicola (unpublished results; Figure 8F).
Figure 8: Epifluorescence (A, B) or confocal (C, D, E, F) imaging of Colletotrichum graminicola expressing fluorescent proteins. (A) Unstained, plasmolyzed leaf sheath 48 hpi with a C. graminicola strain expressing mRFP cytoplasmically under the control of the ToxA promoter34. Significant autofluorescence is also noticed in these leaf sheaths. (B) Unstained leaf sheath 48 hpi with a C. graminicola strain expressing SGFP cytoplasmically under the control of the ToxA promoter. (C)Colletotrichum graminicola expressing SGFP targeted to the endomembrane system with an HDEL anchor35. (D) Unstained leaf sheath 24 hpi with C. graminicola transformed with mRFP under the control of the promoter for the C. graminicola homolog of the BiP chaperone. The red fluorescence in the primary hypha is an indicator of BiP activation in response to secretion stress. (E) Accumulation of arabinofuranosidase::mCherry fusion protein in the C. graminicola WT primary hypha which is crossing the maize leaf sheath cell wall at 44 hpi. (F) Leaf sheath of a transgenic maize line expressing YFP targeted to the plant nuclei30,31, 48 hpi with a strain of C. graminicola expressing mRFP cytoplasmically under the control of the ToxA promoter. Scale bars in each panel are equal to 20 µm, except for panel A where it is equal to 50 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Images in (C-F) were captured with a laser scanning confocal microscope. Please click here to view a larger version of this figure.
Example data 3: Use of maize leaf sheaths to examine the detailed cytology of compatible/incompatible responses and interactions among fungal strains
A non-pathogenic C. graminicola mutant strain (cpr1 MT) was compared to the wild-type (WT) strain in maize leaf sheaths (Figure 9A,B)12.
Live-cell imaging of the strains labeled with cytoplasmic fluorescent proteins demonstrated that the mutant was specifically impaired in movement from cell to cell within the maize leaf sheaths. The sheath protocol enabled precise quantification that revealed that the cpr1 MT was confined to the first colonized cell 95% of the time (Figure 9C)12. This was in marked contrast to the WT, in which less than 5% of the infections remained within the first colonized cell in the same time frame12. Interestingly, the cpr1 MT was able to grow saprophytically beyond the first initially infected cell in locally freeze-killed sheaths, like the WT strain (Figure 9D). This indicated that the inability of the cpr1 MT to colonize was specifically related to an active response of the living maize cell.
The leaf sheath inoculation assay was used to test interactions between the GFP-expressing cpr1 MT and RFP-expressing WT strains by co-inoculating them on the same leaf sheath12. When the strains were inoculated close together (no more than 8 maize cells apart), the cpr1 MT was able to grow normally as a biotroph from cell to cell (Figure 9E,F). Plasmolysis assays confirmed that the cpr1 MT fungus entered living cells12. All these detailed observations were made possible by the sheath assays and implied the existence of a diffusible factor that induces compatibility produced by the WT strain of C. graminicola but lacking in the cpr1 MT12.
Figure 9: Compatible and incompatible interactions involving WT and cpr1 MT strains of Colletotrichum graminicola. Use of different fluorescent proteins allowed the two strains to be distinguished when co-inoculated on maize leaf sheaths12. (A) The WT C. graminicola strain expressing cytoplasmic mRFP in maize leaf sheaths at 48 hpi. (B) The C. graminicola cpr1 MT strain expressing SGFP in maize leaf sheaths at 72 hpi. The cpr1 MT only rarely (<5% of the time) colonizes beyond the first infected cell, even up to 6 days after inoculation. (C) A closer view of the C. graminicola cpr1 MT strain expressing SGFP at 72 dpi. In this case, it managed to cross the cell wall into the next cell, but then it stopped developing. (D) The C. graminicola cpr1 MT strain expressing SGFP, 48 hpi in killed sheath tissues. The cpr1 MT strain colonizes nonliving maize sheath cells rapidly, similar to the WT. (E) When the WT-mRFP C. graminicola and cpr1 MT-GFP C. graminicola strains are co-inoculated close together on maize leaf sheaths (48 hpi), the cpr1 MT-GFP strain regains the ability to progress through living maize sheath cells normally, as a biotroph. Biotrophic growth inside the living cells was confirmed by plasmolysis assays. (F) A closer view of the C. graminicola cpr1 MT strain expressing SGFP, growing from cell-to-cell when inoculated adjacent to the WT (no more than 8 maize cells apart). Scale bars are equal to 50 µm (A, B, and E) or 20 µm (C, D, and F). Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.
Inoculated sheaths have also been used to produce tissues for RNA extraction for transcriptional activity analysis32,33. The sheaths were ideal for this purpose since they could be inspected before extraction to monitor the infection process, thus ensuring sample uniformity across multiple replications. These studies allowed the identification of waves of differentially expressed genes between Colletotrichum lifestyle transition stages32,33.
Example data 4: Results of suboptimal experiments
Unsatisfactory live-cell imaging may be a result of insufficient trimming of leaf sheaths, leading to overlapping epidermal cell layers and optically opaque samples (Figure 10).
Figure 10: Suboptimal experimental outcome due to insufficient trimming of leaf sheaths.(A) Example of multiple epidermal cell layers interfering with the examination of fungal development in vivo. (B) Example of overlapping cell layers affecting live-cell imaging. Scale bars equal to 20 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.
Another potential suboptimal experiment outcome is an unadjusted inoculum concentration that may result in low (Figure 11A) or high numbers of spores: the latter may result in reduced germination or appressorial penetration (Figure 11B). The latter can also hamper the quantification of maize cells colonized at each fungal penetration site.
Figure 11: Suboptimal experiment outcome due to improperly adjusted inoculum concentration. A. Low C. graminicola spore suspension concentration inoculated on sheaths resulting in low numbers of fungal penetration sites. B. High C. graminicola inoculum concentration on leaf sheaths causing multiple appressoria in close proximity, and reduced host cell penetration. Scale bars equal to 20 µm. Micrographs were obtained using a monochromatic digital camera coupled with an epifluorescence microscope. Please click here to view a larger version of this figure.
Time of microscopic observation (hpi) | Developmental stage of C. graminicola inoculated on maize leaf sheaths |
12 | Appressoria |
24 | Primary hyphae |
36 | Secondary hyphae |
48 | Secondary hyphae |
60 | Acervuli |
72 | Acervuli |
108 | Acervuli |
Table 1: Colletotrichum graminicola infection time course: Summary of the time course of maize leaf sheaths inoculated with C. graminicola strain M1.001 based on fungal development milestones throughout lifestyle transition. Microscopic observations were performed every 12 h.
The optimized leaf sheath inoculation method described here is modified from an original protocol that was developed for and has been applied to rice leaf sheaths6,8,36. It allows direct, detailed observations of fungal growth and development in living plant cells with either widefield or confocal microscopy. The protocol is suitable for characterization, comparison, and quantification of a variety of microscopic phenomena during maize colonization, including fungal development and host responses during compatible versus incompatible interactions; production and secretion of specific fungal proteins in planta; and cytology and biochemistry of common or novel pathogenicity factors produced during the maize-fungal interaction. We have used this method with hemibiotrophic and necrotrophic pathogens of maize. We have not attempted inoculations with biotrophic pathogens (e.g., rust fungi) but the sheaths, since they remain alive, would also be useful for that application. We have also applied this method to sorghum sheaths for investigations of the cytology of host and cultivar-specific resistance7,37. For sorghum sheaths, the only adjustment to the protocol was to fill the entire sheath with spore suspension, rather than applying a single drop in the center. This was necessary due to the smaller diameter of the sorghum sheaths.
The use of these sheath assays has greatly advanced our detailed studies on the mechanisms of host colonization and fungal molecules that are critical for pathogenicity. For instance, application of the method demonstrated non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, including hypersensitive resistance responses in both cases7. Maize leaf sheaths were also used to test interactions between fungal strains at a distance on the same sheath. In this case, co-inoculation of a pathogenic WT and non-pathogenic cpr1 MT strain together on living sheaths demonstrated the induction of susceptibility of maize cells by the WT strain and provided evidence for a diffusible factor involved in pathogenicity12. The sheaths allowed differentiation of the two strains expressing different fluorescent proteins, and quantification and statistical comparisons of the colonization process for the strains inoculated either alone or together.
This maize leaf sheath assay has several advantages over whole plant inoculations for live-cell imaging. First, the protocol provides superior imaging, without necessity for clearing or fixation, of pathogens interacting with living host cells in real-time. For instance, studies using maize leaf sheaths enabled the detection of a distinct switch from biotrophy to necrotrophy in hemibiotrophic C. graminicola and facilitated functional comparisons to a non-pathogenic mutant7,12,16,17. Although excised rice leaf sheaths inoculated with M. oryzae reportedly can present symptoms that do not correspond to those observed in whole rice plants36, the timing and appearance of colonization, tissue collapse, and lesion development in maize sheaths is similar to that in whole leaves12,14. A second advantage of the sheath assay is that it shows greater synchronicity across replications and experimental reproducibility of fungal colonization12,18. While whole plant inoculation can result in irregular levels of fungal penetration18, the more highly controlled conditions of the sheath inoculations provide more synchronicity and allow more precise investigation and quantification of interactions. This increased synchronicity facilitated the use of the sheaths in transcriptome analyses32,33. Furthermore, the sheath protocol enabled the necessary speed of sample preparation for transcriptome analysis: trimming, rinsing, and screening each sheath took no more than 2 min before they were flash frozen for RNA extraction32,33.
Critical steps for successful live-cell imaging of infection include: 1) Sheaths should be used immediately after cutting for experiments. Excised inoculated sheaths should not be retained for more than 6 days: if fungal colonization is heavy, they will degrade more quickly12; 2) Be careful to choose sheaths that are of the same developmental age to ensure more reproducible results; 3) Use a fresh spore suspension from cultures that have achieved maximum sporulation; 4) Use sharp razor blades for more efficient trimming to produce optically clear samples; 5) Minimize excessive and unnecessary manipulation of samples as this process can negatively impact live structures and image quality; 6) Check humidity chambers daily to ensure that filter paper is sufficiently moist; and 7) When imaging samples on a confocal microscope, keep acquisition settings fixed during the experiment to avoid introducing intensity changes or bias when comparing fluorescent signals across samples. Fluorescent proteins may photobleach if laser intensity and duration are too high, and they may give weaker signals if tissue sections are kept under a sealed coverslip for a long time. Preliminary experiments, including all appropriate controls, should be performed to standardize and optimize conditions, materials, and timelines for the most reproducible results.
Troubleshooting
Hand-trimming is insufficient to produce optically clear sheaths
It is possible that the razor blade has become dull. If this is the case, dispose of it properly and switch to a new single-edge razor blade. Use moderate pressure and keep the blade at an angle of 90° to the sheath. Scrape the sample gently, but consistently, until observation of a flat and translucent section. It is advisable to check sheaths under a compound microscope before proceeding to the confocal laser scanning microscope.
Inoculated maize sheath is extremely fragile
This occurs when the fungus has colonized most of the plant tissue. Consequently, the tissue is degraded and collapsing. When preparing samples that are already in the necrotrophic stage, gently press the section against a microscope slide, apply a drop of sterile DI water, and scrape the sample one-two times with a clean coverslip.
Low spore germination and penetration in vivo
This may be due to a high number of spores inhibiting germination or penetration. Make sure inoculum concentration is adjusted to 5 x 105 spores/mL. If the issue persists, adjust the concentration to 5 x 104 spores/mL. As a control for the in vivo experiment, place 50 µL of the same spore suspension onto a fresh PDA plate and spread it evenly. Check spore viability and germination daily.
Asynchronous fungal development stages across sheaths
Make sure maize plants are at the same growth stage and sheath sections were obtained from the same node. Also, ensure that spores are viable and fungal cultures are at the optimum stage (e.g., no longer than 2-week-old cultures for C. graminicola) for inoculum preparation.
The authors have nothing to disclose.
The authors thank USDA-NIFA for their financial support (grant numbers 2018-67013-28489 and 2020-70410-32901). Any opinions, findings, conclusions, or recommendations expressed in this manuscript are solely those of the authors and do not necessarily reflect the views of the U.S. Department of Agriculture. We thank Science Without Borders visiting student from Brazil, Mayara de Silva, for the images that appear in Figure 6A and in Figure 7D. We also acknowledge the Department of Plant Pathology at the University of Kentucky for providing access to the Olympus confocal microscopes.
Axiocam monochrome microscope camera | ZEISS | 426560-9010-000 | Compatible with the Axioplan 2 microscope; provides low read noise and high speed for live cell imaging |
Axioplan 2 epifluorescence microscope | ZEISS | N/A | Allows live viewing and image/video capture of biological samples |
Benchtop centrifuge 24 X 1.5/2 mL | Thermo Fisher Scientific | 75002431 | Sorvall Legend Micro 17; max speed: 13,300 rpm (17,000 x g) |
Falcon bacteriological Petri dish with lid | Fisher Scientific | 08-757-105 | Polystyrene material; hydrophobic surface |
Filter paper | Fisher Scientific | 09-920-115 | Whatman grade 1 for Petri plate moist chambers |
FV 3000 laser scanning confocal microscope | Olympus | N/A | For visualization of fungal transformants' |
Germination paper | Anchor Paper Co. | SD7615L | 76# heavy weight for plastic box moist chambers |
Glass Petri dishes | VWR International | 75845-542 | Type 1 class A, 33 expansion borosilicate glass; complete set (cover + bottom), for Petri plate moist chambers |
Glass wool | Ohio Valley Specialty Chemical | 3350 | For glass-wool filter units |
Hemocytometer/Neubauer counting chamber and cover glass | VWR International | 15170-172 | 0.1 mm chamber depth; comes with two 0.4 mm cover glasses |
Microscope coverslips | Fisher Scientific | 12-553-457 | Borosilicate glass; 100/Pk.; 22 mm length, 22 mm width |
Maize cultivar Golden Jubilee seeds | West Coast Seeds Ltd., Delta, BC, Canada | CN361 | Matures in 95-105 days; seed type: F1 |
Microcentrifuge tubes | USA Scientific | 1415-2500 | 1.5 mL capacity |
Microscope slides | Fisher Scientific | 12-550-123 | Superfrost white tab slide; 76 mm length, 25 mm width |
Oatmeal Agar (OA) | VWR International | 255210 | Difco Oatmeal Agar, BD; 500 g |
Nail polish | Revlon | 43671 | Clear nail polish for sealing microscope slides; color 771 Clear |
Non-skirted 96-well PCR plate | USA Sientific | 1402-9500 | 100 uL plate volume |
Pestle for microcentrifuge tubes | USA Scientific | 1415-5390 | Conical tip; polypropylene material |
PlanApo 60X/1,00 WLSM water objective | Olympus | 1-UB933 | Compatible with the Olympus FV 3000 confocal microscope |
Potato Dextrose Agar (PDA) | VWR International | 90000-758 | Difco Potato Dextrose Media, BD; 500 g |
Pro-Mix BX | Premium Horticulture Supply Co. | N/A | Premium general-purpose growing medium formulated to provide a balance of water retention and proper drainage |
SC10 cone-tainers | Greenhouse Megastore | CN-SS-SC-10B | 1.5 inch diameter, 8.25 inch depth, and a volume of 164 mL |
SC10 cone-tainers tray | Greenhouse Megastore | CN-SS-SCTR98 | 24 inch length x 12 inch width x 6.75 inch height; holds up to 98 of SC10 cone-tainers |
Single edge razor blade | Thermo Fisher Scientific | 17-989-145 | AccuTec blade; steel material; 38 mm length blade |
Storage containers/boxes with latch closure | Target | 002-02-0405 | Clear view storage boxes for rmoist chamber; outside dimensions: 23 5/8 inch x 16 3/8 inch x 6 1/2 inch; 32 qt. capacity |