Visualization of Fusarium under Scanning Electron Microscope and Transmission Electron Microscope

Sangya Paudyal

 

INTRODUCTION

The genus Fusarium is a filamentous fungus under the phylum Ascomycota, class Ascomycetes class, order Hypocreales order, while the teleomorphs of Fusarium species are mostly classified in the genus Gibberella, and smaller number of species are classified as Hemanectria and Albonectria genera¹.  According to Leslie and Summerell modern taxonomy of Fusarium, there are 70 species within the genus ¹. Known as the most difficult species to distinguish by pathologists and mycologists worldwide ², the taxonomy of Fusarium is still a continuous debate between lumpers and splitters.

Fusarium genus are well-known mycotoxin producer that act as opportunistic human and animal pathogens as well as a large phytopathogenic genera of microfungi. Many Fusarium species are viewed as weak pathogens that are only capable of attacking plants which have been previously weakened by some other stress factors ³. Found worldwide in all climate zones, soil, plant or plant debris⁴, Fusarium causes diseases like vascular wilt, head and seed blights, stem rots, root and crown rot and canker disease in a wide range of host plants. Out of 101 economically important crop plants of APS list of disease, at least 81 is caused by Fusarium⁵. Species like F. graminearum and F. verticillioides have a narrow host range, and predominantly infect cereals while species like F. oxysporum have a remarkably broad host range, infecting both monocotyledonous and dicotyledonous plants ⁶. The option of control for this pathogen is limited and difficult to implement⁷.

In humans it is known to cause locally invasive diseases like onychomycosis and keratitis in immune normal people as well as infections in persons with seriously burns or those receiving peritoneal dialysis. In immunocompromised people, it is responsible for diseases such as paronychia, invasive sinusitis and pulmonary and extra pulmonary hematogenously disseminated disease with skin lesions and fungemia⁴.  The most frequent species F. solani, F. oxysporum, F. verticillioides (moniliformae) are the major agent of infection ⁴ with F. oxysporum being the emerging pathogen in immunocompromised patients. 

Figure: Classic sickle or canoe shaped macroconidia of 

Fusarium.¹⁰

Fusarium has a distinctive canoe or banana shaped macroconidia, an asexual spore which is the hallmark character of this genus⁸. Spore morphology is the major way of identifying the species of Fusarium ⁹. Macroscopic features of Fusarium include a sclerotium or a sporodochium according to the growth conditions. During favourable condition sclerotium, an organized mass of hyphae, usually dark blue in color is seen. The sporodochium, a cushion-like mat of hyphae bearing conidiophores over its surface, is usually absent in culture. If present, it may be cream to tan or orange color, with an exception for Fusarium solani, whi ch gives blue-green or blue sporodochia. Microscopically, Fusarium consists of hyaline septate hyphae, conidiophores (asexually produced spores), phialides (dilated part of the top of conidiophores), macroconidia (2 or more celled spores), and microconidia (1 celled spore). ¹¹ 

Figure : Ultrastructure of a septate hypha ¹².

Fusarium's hyphae consist of septa with single or multiple pores that divide the hyphae into a series of interconnected hypal compartments ^{12, 13}. Hyphal cells or compartment are either uninucleate or multinucleate. The plasma membrane present in hyphae is closely associated to the rigid hyphal wall. This rigidity that makes it hard to plasmolyse hyphal wall, , is due to the firm attachment of plasma membrane in some regions to the wall. The presence of cytoplasmic organelles decreases with the length of growing hyphae, with the tip having dense cytoplasm and less or no major cytoplasmic organelles ¹². Vacuoles are seen older hyphae in which the biochemical activity is less¹³, sometimes up to the sub-apical hyphal compartments¹².

Fusarium's hyphae consist of septa with single or multiple pores that divide the hyphae into a series of interconnected hypal compartments ^{12, 13}. Hyphal cells or compartment are either uninucleate or multinucleate. The plasma membrane present in hyphae is closely associated to the rigid hyphal wall. This rigidity that makes it hard to plasmolyse hyphal wall, is due to the firm attachment of plasma membrane in some regions to the wall. The presence of cytoplasmic organelles decreases with the length of growing hyphae, with the tip having dense cytoplasm and less or no major cytoplasmic organelles ¹². Vacuoles are seen older hyphae in which the biochemical activity is less¹³, sometimes up to the sub-apical hyphal compartments¹².

Color of the colony, shape and arrangement of microconidia, length and shape of the macroconidia, the number, and presence or absence of chlamydospores together with molecular methods like 28S rRNA gene sequencing can be used for rapid identification to species level¹¹.Spore present in this genus may be conidia produced as simple or polyphialidic slime spores or as enteroblastic spores or as chlamydospores, the spores produced during the resting stage⁹. 
Chlamydospores are the main survival anamorphs and are either formed in the soil or the host tissues and are produced by F. chlamydosporum, F. napiforme, F. oxysporum, F. semitectum, solani, and F. sporotrichoides ^8,9,14.  When present, chlamydospores are in pairs, clump or chains and are thick-walled, hyaline, intercalary or terminal. With the ability to remain dormant in soil and infect other hosts for as long as 30 years, chlamydospores can spread through running water, on farm implements and machinery ¹⁵. With the numbers varying between the genus, chlamydospore formation and number have been widely used as a key structure in systematic nomenclature ¹⁶.

Figure: F. oxysporum’s microconidia 

(2.3-3.5µm)  at various stages of  development

 at the tips of  monophialides (shown

 by arrows). Macroconidia  (3.0- 4.5 µm) are 

the structures with  pointed  apical tips ¹⁷.  

Magnification: ^#1000X+10

Fusarium oxysporum are ubiquitous soil and plant inhabiting microbes, with some specific forms that are plant pathogenic causing wilt and root rot disease over 120 plant species. Causing localized or disseminated infections that may become life-threatening in neutropenic individuals, F. oxysporum is also a human pathogen ^{18, 19}. With production of three types of asexual spores, microconidia, macroconidia, and chlamydospores, the fungus has the ability to survive as mycelium or as any one of these spores. Micronidia are one or two celled and are the spores produced most abundantly and frequently by the fungus under all conditions, even in infected plants. Macroconidia are three to five celled, gradually pointed and curved toward the ends and are commonly found on the surface of plants killed by this pathogen as well as in sporodochia like groups. 

Figure: F. oxysporum’s intercalary 

chlamydospore (5-13 µm) (shown

 by arrow) ¹⁷.  Magnification 

^#1000X+10

Chlamydospores of F. oxysporum are either one or two celled and possess a two-layered wall, the outer layer representing the original hyphal wall and the inner secondary layer formed during maturation of the chlamydospore ²⁰. These are produced either terminally or intercalary on older mycelium or in macroonidia¹⁸.

The cells of hyphae of F. oxysporum are uninucleate with only the nucleus in the apical compartment being mitotically active (Acropetal nuclear pedigree) ²¹. In F. oxysporum, the acceleration of growth between the first and subsequent mitoses has been associated with reduction in hyphal diameter. This differentiation of extremely thin hyphae is relevant to F. oxysporum as it enters its host by direct penetration of the root surface as due to the lack of appressoria, which are swollen tips of hyphae that allow the fungus to directly penetrate the plant tissues, through mechanical and enzymatic activity ^{21, 22}.

The sample used for this study is a Fusarium species believed to have no hyphae. It was obtained from Dr. Andrea Porras lab. The sample was isolated from lichen semiarid soil and was bedded on snakeskin. The genus was identified through the colony characteristics and molecularly confirmed using ITS PCR. Due to the restriction of ITS, the species wasn’t identified at a molecular level, but is believed to be oxysporum according to other characteristics.

MATERIALS AND METHODS:

The Fusarium culture was obtained from Dr. Porras lab and together with the agar, the sample was scooped and placed in 3% glutaraldehyde (0.05M PO₄ buffer, pH 6.8). The sample with the agar was further cut into smaller pieces using a sharp blade.  The cut tissues (together with the agar) was placed in the glutaraldehyde solution for 1.5 hours at room temperature. The sample was washed in 0.05M PO₄ buffer twice and was stored in the buffer in the fridge.

Scanning Electron Microscope:

PO₄ buffer was drained and 35% EtOH(Ethanol) was added in the sample and left for 15 minutes followed by 50% EtOH for 15minutes. The 50% EtOH was removed and the sample was stored in 70% EtOH at room temperature. The sample was dehydrated in 95% EtOH and 100% EtOH for 15 minutes respectively. The sample was then transferred into the specimen basket of the Critical Point Dryer with 100% EtOH. The basket was placed back in the CPD chamber, making sure that the rubber gasket was seated properly. The sample was soaked in liquid CO₂ for a few minutes and the sample was purged to remove the EtOH. Once the paper towel placed on the exit vent stopped detecting EtOH, the temperature was switched to heat until it reached 38^oC. The CO₂ was allowed to escape at the rate of 100psi per minute by slowly opening the bleed valve. The pressure shouldn’t drop in a rate higher than this as the tissue being used may burst. The sample was removed from the CPD chamber. The sample may be stored in dry vials if immediate work isn’t planned. The sample was then mounted onto aluminum studs using silver epoxy. This was done under a dissecting microscope making sure that the fungi sample was facing upwards and the agar (white in appearance) was under the sample. As the samples are really light, extra care was taken not to blow away while placing them under the microscope.

The aluminum stud with the sample was placed in the sputter coater. Extra care was taken when removing the sputter coater chamber, grasping the plastic cylinder rather than the metal top plate. The aluminum stud was placed in the mount holder and screwed. The argon tank was opened after the vacuum dropped below 0.2 Torr.  The leak valve was opened in such a way that vacuum gauge was about 0.5 Torr for ten seconds. The leak valve was closed until the vacuum reached 0.08 Torr. Then the start switch was pressed, followed by high voltage. The leak valve was adjusted in such a way that the Plasma Current gauge read 18 mA. This was maintained for 120 seconds, after which the high voltage automatically turned off. The power switch was turned off together with closing of leak valve and argon tank valve. With the admittance of air to the sputter coater chamber, the aluminum mount was carefully removed. During this process, the sample was coated with gold and palladium layer of around 8 nm thick. The sample was carefully stored avoiding any contact that could knock off the coated layer. Thus prepared sample can be stored for several days in a clean petri plate. On the day for viewing in the SEM at various magnification, the aluminum stud was screwed unto a round disc of brass in the SEM machine. After the placement of specimen the air button was pressed. Once a red light was observed, the High tension that allows enough efflux of the electron required was turned on, followed by WFM. The knob of filament was turned until the maximum output was observed. The WFM was turned off, and using R1 the entire disc was viewed. The area to be focused was determined, and using the magnification knob and focus knob, magnification at 1580X and 400X was performed. R2 was used to focus as it provides better focus. Pictures of desired areas were taken, making sure the contrast and brightness were just right.

Transmission Electron Microscopy

PO₄ buffer was drained and the sample was stained in 2% OsO₄ (0.05M PO₄ buffer, pH 6.8) for 2 hours at room temperature. This was done in a hood as OsO₄ is very toxic and volatile. The sample was then washed in 0.05M PO₄ buffer for several times for 0.5 hr. 35% EtOH was added in the sample and left for 15 minutes followed by 50% EtOH for 15minutes. The 50% EtOH was removed and the sample was stored in 70% EtOH at room temperature. The Spurr Embedding medium was prepared by mixing 5 gm of Epoxy ERL 4221, 13 g of Hardener Nonenyl succinic anhydride (NSA), 3 g of Flexibilizer Diglycidyl ether of polypropylene glycol (D.E.R. 736) and 0.3g of Accelerator Dimethylaminoethanol (DMAE).

The sample was dehydrated in 95% EtOH and 100% EtOH for 15 minutes respectively.  The sample was then mixed with the embedding medium in the ratio 1:1 for one hour followed by mixing in 75% embedding medium for another hour. The solution was completely removed and 100% embedding medium was added and left overnight. The embedded sample was then transferred into an aluminum foil bowl and 100% embedding medium was added till the sample was completely covered. It was placed in a vacuum pump until the bubbles stopped appearing. This was then transferred to a hot air oven at 63^oC, making sure the orientation of the tissue was correct by checking for few times in the first 30 minutes in the oven. This mixture was left for 8 hours in the oven. Glass knives were made using knife maker. Tilted reservoir was made using tape and paraffin (to seal the hole). Extra care should be taken to make sure the knife surface isn’t touched.

The solid disc of embedding medium and sample, was cut into dome shaped rectangular blocks using a jewel saw. The sample block was fixed on a microtome block holder and the top of the block was cut into pyramid shape, with the sample in the middle of the pyramid roof, using sharp blades under the binocular microscope. Once the desired shape was attained, the block holder with the block was placed on the ultra-microtome. The knife was placed in a knife holder at 5^o. Water was filled in the reservoir, leveled to the knife edge, giving maximum reflection. The knife was adjusted in such a way that it was in plane with the sample block. The microtome was turned on and the sample surface was made smooth. Then few thick sections were cut. These thick sections were stained with Toluidine blue and viewed under light microscope. This allows us to know if there is sample in our block. The temperature was increased in the microtome, to get thin sections. Sections of correct thickness appears silver or pale gold under the microscope. Once these section start appearing, the copper grid was cleaned first by mashing it with hands, followed by immersing it in EtOH, Hydrochloric Acid and Water. Extra care should be taken to make sure the copper grids are dry. Using toothpick with an eyelash, the unwanted sections were moved. The copper grid was then placed in the area with desired sections and transferred to a petri plate with filter paper. Thus prepared sample was left to dry.

The copper grids were stained in 2% Uranyl Acetate solution for 10 minutes followed by staining in Lead Citrate for 5 minutes and immersed in water to prevent formation of lead carbonate crystals. The grid was transferred to the TEM and pictures were taken at various magnification. The negatives were developed by immersing the film in Developer for 3 minutes, followed by immersion in water for 30 seconds and fixative for 7 minutes followed by water for 20 minutes. The negatives were allowed to dry and placed in envelope. The magnification and image number were noted in the envelope.

For printing, the negative was kept on a negative holder and inserted into the camera. Printing paper was placed on the easel holder with the emulsion side up. This was all done in red light.  The printing paper was exposed for 30 seconds and transferred to the developer and immersed till the image started appearing. Then the photographs were transferred to a stop bath (5% acetic acid) for 2 minutes followed by immersion in fixer for 15 minutes. The photographs were then immersed in water for 30 minutes. The Drier drum was used to dry the photographs.

RESULT:

Scanning Electron Microscopy: A big mass of hyphae was observed together with conidia and chlamydospore at 400X and 1580X magnification. Figure 1 shows the SEM picture of the Fusarium at 400X. “a” shows the region of hyphae with conidia and “b” represents the area that was further magnified. In figure 2, a chlamydospore can be seen sitting on top of the mass of hyphae, represented by “A”.

Transmission Electron Microscopy

Various sections of hyphae was observed in the TEM at a magnification of 27,000 and 35,000X magnification. Figure 4 shows hyphal wall and plasma membrane at 35000X Magnification. It appears to be a transverse section of hyphae. Figure 4 shows a cross-section of hyphae of about 1.5-2 µm diameter. Cellular structure such as nucleus, nucleolus and lipid droplets are visible.

Figure 3: Transmission Electron Micrographs 

of Fusarium at 35,000X magnification. 

Hyphal wall “i”, plasma membrane  “ii” and 

septa “iii” are visible.

Figure 4: Transmission Electron Micrographs

of  Fusarium at 27,000X magnification.

Hyphal cell (1.5- 2 µm diameter) seen.

Visible Nucleus (N) and  nucleolus

 (n) together with lipid droplets (L).

DISCUSSION AND CONCLUSION

Fusarium, a filamentous fungus widely distributed on plants, in the soil and in water, is a plant pathogen as well as a causative agent of superficial, locally invasive and systemic infections in humans.  The Fusarium under study was believed to be devoid of hyphae, rendering it as an unhealthy sample.

Figure: SEM analysis of healthy hyphae of 

Fusarium graminaerum on PDA medium at

5^th day after incubation at 28°C. ²⁰

In 400X, a big mass of hyphae was observed. Conidia on the hyphae were also observed. Compared to the SEM picture of a healthy hyphae in a research work on the effect of Bacillus subtilis on Fusarium graminaerum, with hyphae about 1.2 µm- 4.8 µm in diameter ²⁰, the hyphae seen in the SEM micrograph of Fusarium had the same diameters ( 2.5- 3.2 µm). The round structure seen under 1580X is a chlamydospore with a diameter of about 8-10 µm. Compared to the literature found, the chlamydospore had similar dimensions to those of healthy ones (5- 13µm ¹⁶).

Figure 3 appears to be an angular transverse section of the hyphae. First I thought it was a spore but when measured it was too big (10 -12µm) to be a microconidia as well macroconidia.  The presence of only 1 nucleus and no cell wall within the structure also refutes the idea of it being a macroconidia or chlamydospore. The plasma membrane and hyphal wall are clearly seen in the TEM photograph. As the specimen seems to have been cut at an angle, incomplete septa are also visible.   

Figure: TEM analysis of Fusarium oxysporum

hyphae inside the epidermal cell wall as 

well as inside an epidermal cell at 72h of 

Orobanche tubercles. ²³

Figure 4 is a cross section of the hyphae. Its uninucleated and nucleolus is visible. Lipid droplets ²³ are seen in the cytoplasm. The cell is about 1.5- 2 µm in diameter, with similar structure to the TEM analysis of F. oxysporum infection in Orobanche tubercules. The difference in cell diameter can be accounted to difference in cell size between different lengths of hyphae, with cells near the tip having smaller diameter ²¹.

Figure: F. oxysporum: 1000X ¹⁷

The sample contained hyphae, despite the assumption that hyphae was absent. According to the classification of Fusarium based on spore morphology, F. oxysporum consists of one or two celled spores known as microconidia, a spore most abundantly and frequently produced this species. The conidia observed in the SEM pictures were somewhat similar to conidia seen in the picture of F. oxysporum 1000X ¹⁷. Similarity in structure in the TEM pictures between my sample and the F. oxysporum in the literature, also helps to establish that that the sample was indeed a F. oxysporum.   

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