Critical point drying

Groundnut cultivars UF 71513 and TMV2 were infected with A.

parasiticus, I MI no. 120920 and a second batch of TMV2 seed was

infected with A. flavus, IMI no. 93803, as described in Chapter Two 2.2.1. Pieces (4mm x 5mm) of groundnut 10 days after inoculation with Aspergillus spp. or uninfected testa (cv. Ill) were cut and dehydrated through a graded series of aqueous alcohol starting with 5% alcohol (ABS) and 10% ABS as the second step followed by 10 % increases of ABS to 100% ABS. The samples were washed in each solution before immersion for 30 mins. Samples were kept in 100% ABS before drying with a Bio Rad critical point drier.

6.3 RESULTS

6. 3. 1 Fixation with osmium tetroxide

Fixation of the mycelium resulted in the drying of the agar pieces as manifested by their darkening and hardening. The best quality samples were obtained from the edges of 3 day old colonies. Vesicles, phialides, sterigmata, conidial heads and conidia retained their turgidity while in contrast, mature mycelium always collapsed (Figs. 6.1-6.5).

Comparisons between the two Aspergillus species revealed differences in vesicles and spore formation (Fig. 6.1, 6.5 ). The vesicle shape of A. flavus is ovoid and that of A, parasiticus

flask-shaped. Conidia are formed by being 'blown out' from flask­ shaped cells termed phialides which are subtended by the vesicle

(Fig. 6.5). Under light microscopy differences between isolates of

A. parasiticus and A. flavus cannot be discerned. However, using

SEM, spore topography demonstrated that A. flavus has a verrucose ornamentation, ie. wartlike out growths on the surface (Fig. 6.4,

and A. parasiticus^ an echinulate or spinelike ornamentation (Fig.

6. 3).

6. 3. 2 Air drying

Shrinkage of conidia onto the supporting medium was highlighted with air drying for 24 h (Fig. 6.6 b and d). Consequently it was difficult, in some instances, to judge ornamentation of conidial walls. However, samples air dried for 12 h showed little or no shrinkage (Fig, 6.6 a and c), It was possible in most cases to

distinguish between isolates- of A. flavus with its verrucose pattern and A. parasiticus with its echinulate pattern.

6,3.3 Critical point drying

Scanning electron micrographs of infected groundnuts provided a general picture of infection (Fig. 6.7 a). The infected groundnut exhibited a superficial layer of fungus in various states of growth and reproduction, as well as the testa and the cotyledon. Mycelia and fruiting bodies were evident and shrinkage was also visible (Fig. 6.7 b and 6.8 a). It was difficult to see clear proof of penetration of the hyphae into the testa (Figs. 6.7 b and 6.8 b).

Cross-sections of uninfected testa were also examined from groundnut cv. 111. The testa seen in Fig. 6.9 a is representative of a batch of seed tested on receipt from India and was subsequently found to behave characteristically as an IVSCAF resistant cultivar where fungal growth was limited and phytoalexin production high (data not shown). The testa seen in Fig. 6.9 b is representative of a batch of 111 seed that had been stored for 9 months and had demonstrated IVSCAF susceptibility; fungal growth was considerable and phytoalexin concentrations low (Chapter Four section 4.3.5 and 4.4). On comparison (b) had a thinner inner parenchyma layer due primarily to reduced cell wall thickness. The middle parenchyma layer lacked a spongy appearence near the inner parenchyma layer and a fluffy appearence near the outer sclerenchyma layer. The outer sclerenchyma layer appeared less dense than that of 111 (a).

6.4 DISCUSSION

Weidenbôrner et al. (1989) reported the ease with which osmium tetroxide fumes could be used for the preparation of Aspergillus

spp. for scanning electron microscopy. Kozakiewicz (1982) described air-drying as a method of preparation to judge ornamentation of conidial walls. In these experiments fixation in osmium tetroxide vapours was the method of choice to study the structures of the

Aspergillus flavus group of fungi. Conventional methods are

laborious and time consuming. These include fixation, washing, dehydration and critical point drying (Brown and Brotzmann 1979, Cole and Samson 1979). Fixation in osmium tetroxide vapours was carried out by Dowsett et al (1977) for studying Dactylaria

brochopaga, by Quattlebaum and Carner (1980) for preparing

Beauveria spp. and by Elad et al (1987) for observing the

interaction between Trichoderma spp. and Rhizotonla solani.

In order to prevent shrinkage of structures only young mycelium should be used to study this genus by SEM. Old mycelia are not suitable for fixation in osmium tetroxide fumes as they readily collapse (data not shown). This phenomenon seems to hold irrespective of whether they were fixed by air drying or critical point drying. Tokunga et al (1973) suggested that the collapse of mycelium was caused by the protocols of conventional fixation and dehydration, j

I

However, mycelial collapse was evident in this study even with fixation in osmium tetroxide fumes where there was no physical contact between the fixatives and the fungi (Fig. 6.4

b). Therefore, contact with fixatives and dehydration solvents such as absolute alcohol seem not to be responsible for the collapse. A more probable candidate is age of the mycelium.

Specimens prepared with osmium tetroxide possessed good electron conductivity and overall produced good micrographs.

Air dried samples used to study the ornamentation of conidia were subject to shrinkage if the samples were exposed to the air for long periods, ie. 24 hours. The method itself was easy and quick to follow and did prove to be useful in identifying isolates of the Aspergillus species. It has been stated that there is no sharp line of demarcation between A. flavus and A. parasiticus.

Differentiation of these two species depends on colour, conidiophore length and conidial ornamentation. Kozakiewicz (1982) has highligted the fact that although SEM has improved the understanding of Aspergillus taxonomy, it has introduced an additional problem - the extreme changes in external morphology exhibited by the conidium during ontogenetic development. She illustrated the development of the initially smooth conidium of A.

nigen through lobate, striate to an ultimately verrucolose

condition. When using the proposed time saving technigue of in situ examination, selection of any one of these stages other than the verruculose mature form may lead to misidentification of the species. This is clearly demonstrated with the conidia of A.

asperescens IMI 46813, an example of an Aspergillus Isolate where

immature forms differ markedly from the mature state; the immature condition equates with a mature form found in other species groups

Critical point drying was effective in sample preparation. Although shrinkage was evident an overall picture of the infection of groundnut seed was visible as was the structure of the testa. The actual infection process was not evident. The fungus was seen growing on the groundnut testa but evidence of fungal penetration through the testa into the cotyledon was extremely difficult to find. To study the histology of groundnut infection by Aspergillus

spp. thoroughly, Transmission Electron Microscopy should be used. Comparisons of the two testa types of 111 suggest that the testa was somehow damaged in batch <b), possibly by rapid drying of the groundnuts (Fig. 6. 9 a and b). The diversity among groundnut cultivars relative to maintenance of seed quality during accelerated drying or fluctuations in drying rate needs to studied (see Chapter Four section 4.4). The identification of structures in testae vhich may be associated with seed quality need to be ascertained.

In conclusion, fixation in osmium tetroxide fumes allowed fungal structures to be studied. Air dried samples were useful in distinguishing the conidial ornamentation differences of A.

parasiticus and A. flavus. SEM proved not to be a suitable

technique to study the histology of groundnut infection by

Fig. 6. 1.

Two young conidial heads of Aspergillus parasiticus var.

globus Murakami IMI no. 120920 bearing spores (1). Swollen vesicles

are also visible but these were too young to produce conidia (2). Hyphae are visible in the background (3). The specimen was prepared by fixation in osmium tetroxide fumes.