Food Structure

Volume 2 Number 2 Article 4

1983

Infection of Oriental Mustard by Nematospora: A Fluorescence and Scanning Electron Microscope Study

Richard A. Holley

Beverley E. Phipps-Todd

Suk H. Yiu

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Recommended Citation Holley, Richard A.; Phipps-Todd, Beverley E.; and Yiu, Suk H. (1983) "Infection of Oriental Mustard by Nematospora: A Fluorescence and Scanning Electron Microscope Study," Food Structure: Vol. 2 : No. 2 , Article 4. Available at: https://digitalcommons.usu.edu/foodmicrostructure/vol2/iss2/4

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INFECTION OF ORIENTAL MUSTARD BY NEMATOSPORA: A FLUORESCENCE ANO SCANNING ELECTRON MICROSCOPE STUDY

Food Research Institute Research Branch Agriculture Canada Ottawa Ontario, Canada KlA OC6

Introduction

Fluorescence light microscopy and scanning It is not uncommon to find that spices are electron microscopy were used to study penetration naturally contaminated with bacteria and fungi by the yeast Nematospora caryl i through the seed (Chandra et al. 1981, Ayres et al. 1980), to such coat and into the embryonic tissues of orient al an extent th~in many countrTeS1hei r steri l i za­ mustard seed {Brasslca iuncea). tion by treatment with ethylene oxide is routinely Infection of the seed was associated with its carried out. This is done mainly to reduce the phys i cal injury; however, it was evident that the risk of pathogenesis as well as ea rl y food spoil­ yeast was capable of successfully invading healthy age by the introduction of large numbers of micro­ plant cells. The pathological process was fol­ organisms during seasoning. Under normal circum­ lowed in parallel using both the above types of stances most of the organisms present in spice mi croscopy. Foci of yeast infection on the seed seeds (~.!. ..9..=..• anise, caraway, celery, coriander, coat outer surface were characterized by swell ing cumin, nutmeg, dill, fennel, mustard, poppy, pep­ of the infected epidermal cells. Nematospora hy­ per , sesame) are on the surface of the seeds phae were seen in the 1 umi na of the seed coat (Cowlen and Marshall 1982, Leistner ~~1981, pa 1i sa de ce 11 s and spread 1atera 11 y when the hya- Pivnick 1980). Indeed, Leistner found that below 1i ne 1 ayer between the seed coat and embryo was the palisade layer of the peppercorn testa, the reached. Sites of infection at the surface of seed was essentially sterile. It should be noted cotyledon ce ll s appeared as zones of localized that Chandra et al. (1981) found about 25~ of erosion. Asci and spores were visible, embedded seeds to be stffi Wected following surface dis­ in disorganized and disintegrating plant tissue. infection, although this may reflect storage at higher than normal humidity (Schans ~ ~ 1982). In spices several compounds, but, in part icu­ lar, essential oils(~. isothiocyanates), are potent antimicrobial agents {Vlrtanen 1962 , Piv­ nick 1980). It is believed that this is also true in oriental mustard with respect to Nematospora £.2.!:.Yl.!. (Holley and Tim~e:s 1983). However, de­ spite the natural tox1c1ty of the seed to the infecting Nematospora it became of some interest Initial paper received August 23, 1983. to examine the development of yeast ~enetration Final manuscript received November 11, 1983. into the seed, especially since the latter occur­ Direct inquiries to R.A . Holley. red spontaneously in the field. Telephone number: 613-995-5265 . Nematospora coryli is an internationally im­ portant plant pathogen capable of causing devas ­ tating damage to many crops in different parts of the world. Phytopathogenic Nematosporaceae are more frequently found in warmer parts of the world (Batra 1973) but recently were reported to occur in oriental mustard grown in a restricted area of western Canada (Burgess et al. 1983). Although at present the outbreak ha-5 abated, it has become important to examine the development of yeast penetration i nto the seed, especially in view of the expanding role mustard crops will probably KEY WORDS: Ascospore morp hol ogy; Fluorescence play in Canad ian agriculture. Concern is expres­ ililcroscopy; Mustard; Nematospo ra; Plant pathogen; sed that crop quantities larger than 80,000 hec­ Sca nning electron microscopy; Seed morphol ogy; tares planted to mustard in Canada in 1982 may be Sp i ce ; Yeast infection. at risk. In addition, should mustard serve as a

143 R.A. llOLLEY 1 B . E . PHIPPS - TODD a nd S . H. YIU reservoir f o r infection of other susceptibl e Fluorescence microscopy crops, the potential for damage would be s i gnifi­ Mustard seeds were fixed and embedded i n cant. glycol methacry l ate ( G~lA)(Eastman Kodak Co., Ro ­ chester , NY) us i ng the method described by Y; u et al. (1982 ). Briefly, seed tissues were fixed in 3% gT"Utaraldehyde in 0. 025 M potassium phosphate Electron microscopy buffer, pH 7.2, at 4"C for 48h, dehydrated through Oriental mustard seed (Brass ica juncea) for methyl cellosolve , ethanol, n-propanol and n­ these studies was obtained from a pocket of infes­ butanol, and infiltrated with GMA for 3 to 5 daYs tation in a field in southwestern Saskatchewan. prior to polymerization at 60°C in gelatin cap ­ In order to ensure that individual seeds for mi­ sules. Sections were cut 3 to 7 1-1m thick using croscopi c examination were infected with Nemato­ glass knives and affixed to glass slides for ex­ spora, they were dissected approx i mately in half amination. with a scalpel. One half was stored for later Mustard seed sections were stained with 0.05% microscopic examination, the other was crushed in (w/v) aqueous Aniline Blue (C.!. 42755 , Polysci ­ a sterile mortar with a pestle. The crushed seed e~ce Inc. , Warrington, PA) in 0.07 M K3 Po 4 for 1 was mixed with molten 50°C plate count agar (Dif­ m1n and/or 0. 001% (w/v) aqueous Cal cafruor White co) containing 50 ppm each of tetracycline and M2R (American Cya na mide Co ., Bound Brook , NJ) for chloramphenicol to retard bacterial development. 1 to 2 min. After a rinse in water t hey were air­ These additions of antibiotics , lower than the 100 dried , mounted in immersion oil, and examined for ppm recomme nded (Speck 1976), were used because fluorescence using an exciter/barrier filter set Nematospora growth was retarded by higher concen­ with maximum transmission at 365 nm/>418 nm (FC I ) trations. The seed- agar mixture was allowed to or at 450 - 490 nm/>520 nm ( FC II ). Alternately, solid i fy in a petri dish and incubated at 35"C seed sections were stained 2 to 5 min in 0.01% under oxygen-free nitrogen for 4 to 5 days to (w/v) aqueous Congo Red (C . I. 22120, Fisher Scien­ further reduce competition from bacteria and fila ­ tific Co., Fairlawn, NJ). They were then rinsed mentous fungi. The presence of typical subsurface in water, air- dried, and mounted in non-fl uores­ cream - coloured and star-shaped colonies in the cent immersion oil for fluorescence examination agar was taken to be indicative of Nematospora and using either FC II or an exciter/barrier filter this was confirmed by phase contrast light micro­ set with maximum transmission at 546 nm/>590 nm scopy. Approximately 20% of seeds examined from ( FC II I) . this source were contaminated. Visual inspection All sections were examined with a Zeiss Uni ­ and separation of mec hanically damaged seeds was versal Research Microscope (Carl Zeiss Ltd., Mont­ not an efficient or productive method for i sola­ real, Quebec) equipped with a III RS ep i-il lumi­ tion of infected seeds. For comparative purposes , nating condenser combined with an HBO 200 W mercu­ our original isolate _of Nematospora cor~li (Holley ry- arc il luminator for fluorescence analysis. The and Timbers 1983) wh1ch had been lyophi ized , was I I I RS condenser contained a 11 three fluorescence used as a reference in pure culture work. filter combinations of FC I , 11, and III. Photo­ When it was determined from incubation of the micrographs were obtained using 35-mm Kodak Ekta­ halves of suspect seeds in agar that seeds were chrome 400 ASA daylight fi 1m. infected with Nematospora, the other half of the seed was bisected. Seed samples were fixed for 24 h at 4 °C in 2.8% gl utara 1 dehyde. Fixed seed was rinsed three times in distilled water, fro zen in Scanning electron microscopy melting freon, transferred to liquid nitrogen, Seed coat Normal uninfected mu stard seed as freeze - fractured , and then freeze-dried at · 80°C viewedTrDmthe outer surface looked very much (S peedivac- Pearce Tissue Dryer Model 1). Frac­ like a golf ball. The surface contained a semi­ tured seeds were mounted on aluminum stubs with regular array of ridges which compartmented the silver cement , and coated with carbon and gold (20 seed coa t to form a network that gave an almost nm) in a coating unit (Speedivac Model 12E6/1258, honeycomb pattern (Figs. 1 and 2) with little Edwards High Vacuum Limited, Crawley, Sussex , Eng­ apparent debris and no significant interruption of land). Specimens were examined in a Cambridge the surface pattern. Rarely, an ascospore could Stereoscan Mark 2A scanning electron microscope be seen on the outside surface of a normally (SEM) at 20 kV. appearing seed coat of an infected seed (Fig. 3). Pure cultures of Nematospora grown on plate Examination of Nematospora pure cultures by count agar (O ifco) at 35°C for 3 to 7 days were SEM revealed that as the culture aged beyond a removed from the agar surface and fixed in 2. 8% week , hyphae and spindle-shaped ascospores predom­ glutaraldehyde for 4 h at 4°( in a test tube. The inated (F ig. 4}. These latter ones were seen fixed cultur e, now a suspension, was transferred together with elliptical vegetctive cells in in­ to freshly cleaved mica sheets (5 x 13 mm) which fected seed tissue. had been pretreated with 0.1% aqueous poly- L­ When seeds known to be infected by the yeast lysine hydrobromide for 20 min and rinsed with were examined further, most of the seed surface water. Samples were applied before the mica appeared to have the normal gr id- like pattern; dri ed. After 15 min exposure on the mica support, however, areas where this patte"n was interrupted samples were rinsed in di st i 11 ed water three were visible at intervals on t11e seed coat (F ig. times, frozen in melting freon , transferred to 5} . Interruptions consisted of raised , somewhat liquid nitrogen, and freeze- dried. Samples were smooth areas which were "pebbled" and appeared as mounted on SEM stubs and treated as described if they were areas of seed epidermis swollen by previously for SEM examination of seed. the growth of an underlying y~ast microcolony.

144 ORIENTAL MUSTARD INFECTION BY NEMATOSPORA Globose and ell i ptical vegetative yeast cell s were visl bl e in the vicinity of these affected areas on the seed surface (Fig. 6). The contents of the raised areas were not a mo rphous as wou ld be ex ­ pected if i t were mucilage. Further examination revealed that these raised areas contained a tightly pac ked array of ell i ptical and globose yeast cells (F ig. 7). I t was usual to find sites of what appeared to lbe physical injury near where swollen yeast ­ infected tissue was seen at the surface of the see·d c oat (F i g.6). Visible lesions were not clearly defined on the inside surface of the seed coat since this surface did not fracture cleanly. Often amorphous str in9y debris would adhere to the exposed sur­ face. Most spectacularly and in association with many infected cells, the hyaline layer was filled with vegetative yeast cells (Fig. 8). Occ as ional­ ly rn at ure asci and ascospores were also vi sible. Cotyledon surface Cotyledon tissue wa s also affected by the development of the pathogenic Nem atospora. Zones of eroded or part i a 11 y digested tissue were evident in isolated areas across this surface and occurred only where the infecting yeast was present (Fig. 9}. Frequently, evidence of extensive tissue damage due possibly to physi ­ cal injury was present in the areas of the lesion (F i 9. 10). App arently undamaged coty ledon cell s were also infected by the invading yeast. The presence of spores in these cells and t he deve 1op ­ ment t oward disor ganization of seed cell structure was also seen (Fig. ll). Fl worescence microscopy When a smear containing a 6- day old broth culture of Nematospora was prepared on a glass slide and was dried and stained with Aniline 13 lue , the r esults obtained were as shown in Fi g. 12. Gl obose and el liptical cells we re seen to have folds in their cell wall s which fl uo resced a pale green co l our. The ascospores were also stained fl uorescent, but with i ntensi ty at both the tip of the anteri or (acumi nate) end and the enti re poste­ rio ~r half of the spo re. No fluorescence was noted in the mi d- to anterior region of the spore. It has been reported that Aniline Blue dye is specif­ i c for a- (l - 3)- 0- gluca ns (Ful crer 1982) wh i ch almost certa inly occu r in t he Yegetative cell walls and those of spores. Ho wev Er , the possibil­ ity that the dye may have affinity for other chemi ca l groups cannot be ruled out . Certa inly, the r e sult obtai ned here refl ec:s a difference bet~Ween the anterior and postericr halves of the spo~res . Differential staining of t hese spores has been reported by others, with the anterior portion being refractile to staining with Ac id Fast and cytoplasmic stains (Carmo- Sousa 1970 , Batra 1973). tu. n examination of GMA - embecded serial sec­ tions of infected seed halves show:d results simi - 1 ar to those found during SEM . Yeast on the

Fig .. 1 . Surface view of an uninfected oriental must:ard seed by SEM. Fig. 3 . Su rface view o f the seed coat from an Fig~ 2 . Seed coat of oriental nustard by SEM , infected mustard seed showing a single ascospore showing semiregular network of su1face ridges. (ar row) of Nematospora.

145 R . A. HOLLEY , B.E. PHIPPS - TODD and S . H . YIU

Fig. 4, Hyphae and pseudomyceliur.t of Nematoseor a Fig. 6 , seed coat surface of an infected seed coryli also showing ascospores (arrow). showing exposed yeast ( arrow) and a swollen area of the seed surface where physical inj ury ( double arrow) may have been infl icted.

Fig. 5, Surface view of the seed coat from an Fig . 7. Seed coat surface of an infected seed infected mu stard seed showing interruptions (5) showing an area where subsurface yeast growth has in normal sur face pattern which are believed to be caused swelling (S), vegetat ive yeast cells can be areas of Nematoseora involve ment. seen below the seed coat surface ( arrow),

outside of the seed was integrated among cells of epiderm i s i s shown in Fig. 16. Significant damage the muc i lagenous epidermal layer (Fig. 13} . This wa s seen be neath the aleurone cel ls while the growth by the yeast mycelial form was observed to aleurone laye r appeared largely unaffected. Yeast penetrate through the subepidermis into the pal i ­ spores were seen t o spread laterally throughout sade cells where yeast were visible in the lumi na the hya 1 i ne 1 ayer and in some preparations actua 1- of the pa l isade cells (F ig. 14) . A zone of eroded ly circled the entire ·em bryo. Tissue damage was tissue or a site of physical injury was also also vi sible in the peripheral cells of the coty­ vi sible. A cross sectional view of the seed coat ledon (Fig. 17} and yeast spores with their char­ is shown in Fig. 15. Fungal hyphae are visible in acteristic arrangement in pa c kets of eight were the mucilagenous epidermal tissue. Extens i ve da­ seen in cross- section inside some of the infected mage can be seen in the pigment layer above the cotyl edonous cells (F i g. 18 ). No infection was aleurone cells as wel l as in the latter tissue. detected beyond the periphery of the cotyl edonous A cross- section of the seed coat below the tissue.

146 ORIENTAL i'1USTARD INFECTION BY NEMATOSPOR A

Fig. B. 4. fractu r e of an infected orient al mustard Fig. 10. Cotyledon surface from an infected seed seed tagential through the aleur one layer of the illustrating t i ssue disorganization resulting from seed cat (A) through the hyaline laye r showing Nematospora infection from a site of physical vegetative yeast cells ( Y ) in a matrix ( M) of injury (I) . Yea st asci (arrows) are visible in amorphos mate r ial . necrotic tissue.

Fig, 9. View of the cotyledon surface of a n in­ Fig. 11. Yeast ascospores ( arr o w) " exuding" from a fected 1us tard seed showing the progression of cel l of the surface of co t yledon tissue in an Nemato.pora infection of healtily tissue . No rmal infected seed. Other adjacent cells appear d is­ h ea l th1 cells ( C ), stressed cells ( S) , amor phous torted perhaps due to the yeast infection. materi~ (M), and yeast cells (Y) are visible.

Discussion ture of the yeast was its spindle- shaped asco­ spores which possessed spiral ridges on the poi nt­ Gr c<~ th of Nematospora in pathologica l lesions ed or posterior end which resemb led an auger (Fig. of the !:!ed or in laboratory culture resulted in 4). In all probability this pattern on the sur­ the sa lT!' diversity of cellular mo rphology. The face of the spores was noth in g mo re than the distinctcharacter of vegetative cells, mycelium, decoration that has been reported before on funga 1 and ascc pores was mai ntained in the two environ­ spores (M artinez~~ 19 82). The ridges may ments ad equivalent yeast forms were observed in serve to some extent in the process of spore each miieu. The most striking morphological fea- dissemination .

147 R. A. HOLLEY, B . E . PHIPPS- TODD and S .H. YIU

Seed coat - ex terna 1 Images obtained in cross sections of the seed --When-seed rinse and surface sterilization coat (Figs. 15 and 16) were identica l in outline procedures (Chandra et al. 1981) using 2'X. sodium to those previously published for .!h._ iuncea (Aoba hypochlorite were used;-Tlttle evidence was ob­ 1972, Vaughan et al. 1963) by 1 ight microscopy and tained for the presence of contaminating Nemato­ were simi l ar tOthose pub l ished for yel l ow mustard spora on the seed surface. An examination of ( Vaughan~~ 1g76) and for rapeseed using fl uo­ contaminated seed by SH1 and fluorescent light rescence microscopy {Fulcher 1982, Sc hans ~~ microscopy did result in the observation of sur­ 1982, Yiu et al. 1982). In cross section, three face contamination on the seed coat, usually, but major layer"SoTCells were evident in the oriental not always, adjacent to foci of epidermal infec­ mustard seed coat: the outer epidermal, underlying tion (Figs. 3, 6 , and 13). The proportion of palisade layer, and inner aleurone cells (F ig. organisms on the seed surface easily removed by 15). The hyaline layer between the seed coat and surface rin·sing was small in relation to the total cotyledon cells was also visible (Fig. 16) but numbers of organisms present in infected seed detail of parenchymal tissue overly ing the aleu­ {approx. 1%) , although for surface-contaminated rone cells was not clear in infected specimens. seed rinse-soak methods are recommended to rou­ Yeast and mycelia were present on the outer tinel y quantify microorganisms (Cowl en and Mar­ epidermal layer (Figs. 6 and 13) and were believed shal l 1982). to penetrate into and through the lumina of the Oriental mustard seed surface topography palisade cells (Fig. 14) , the aleurone layer (Fig. {Figs. 1 and 2) resembled in a very general way 15) and then to the underlying hyali ne layers images of 8 rassica ~ published elseWhere (von where lateral spread and multiplication of organ­ Hofsten 1g74). Brassica nigra (Vaughan ~ .!!.,_ isms occurred (Fig. 16). Shown in Fig. 8 is a 1976), and also black pepper (Leistner et al. comparable view by SOl , tangential to the hyal ine 1981) with differences noted in the folfOw:rng layer through the aleurone layer. Vegetat i ve discussion. The predominating feature of the seed cells of Nematospora were vis ibl e in large num­ coat appearance was an informally arranged network bers. Often by both SEM and fluorescence micro­ of interconnecting ridges (F ig. 1). There were scopy the hyaline layer was seen to be filled with fewer ridges on the seed coat of b 1 ack pepper than both vegetative cells (SEM) and spores {fluores­ on oriental mustard. On~~ surface ridges cence). were closer together and valleys in between were Cot y 1edon penetration deeper. With~· ni gra the same patte_rn was evi ­ Cotyledon tissue was heavily infected in some dent but the r""l""dges were not as consp1cuous (Vau­ peripheral areas wi_th localized foci of eroded ~nd ghan et al. 1976) and the surface was more 1 ike apparently necrot1c tissue often in assoc iat1on that Of arT ental mustard as seen by fluorescence with vegetative yeast cells {Fig. 9) . In contrast microscopy (Fig. 13). with the swollen areas on the seed coat surface , Surface contamination of oriental mustard by these erosion zones (Fig. 10) resembled erosion Nematos ora was visible by both SEM {Figs. 3 and troughs around the bacterium Alteromonas putrefa­ 6 and fluorescence microscopy {Fig. 13), and ciens on pork skin {Butler ~E..1_ 1 980). Results occurred mainly in areas where physical damage of were interpreted to mean that foci of yeast infec­ the epidermis was visible {F i g. 6). If the latter tion developed in areas that had suffered physical were "puncture" damage , in all probablllty this injury, although the yeast appeared to be an inva­ physical injury was due to the feeding activity of s ive parasite. For example, amorphous tissue was insects like the false chinch bug (Nysius erlcae) often found in an area adjacent to a focus of and others which have been implicated as vectors infection. As one moved farther from the focus of in disease transmission (Burgess et al. 1983, infecti on , intact cotyledon cells could be seen Heinriclls et al. 1g76, Batra 1973). Prlor--physical which contained structures resemb ling maturing injury i s COnsidered to be an important IJrerequi­ ascospores {Fig. 9) . Thus, Nematospora appeared site for the establishment of infection in spice capab 1e of infecting otherwise normal tissue - an seeds (Leistner et al. 1981). observation made by Heinrichs~ .£.l.=._ (1976) during Foci of NemaiOSOora infection on the outer a study of inocula ted soybeans. seed coat surface appeared as elevated or swollen The pattern of oriental mustard seed infec­ areas and interrupted the normal pattern of sur­ tion by Nematospora seen he re at each layer of face ridges {Fig. 5). These elevated areas had a tissue seemed to be associated wit 'l physical inju­ "pebbled" appearance due to the underlying masses ry, and was likely caused by an insect vector of globose and elliptic al vegetative cells (Fig. (Burgess ~ .!!.,_ 1983). There appears to be a 7). Engorged areas probably developed as a result consensus that physical injury, probably through of rapid yeast growth prior to seed maturation and insect feeding with consequent Nenatospora inocu­ desiccation in the seed pod. This hypothesis is lation of the damaged seed, is a necessary pre­ consistent with the result obtained by Burgess et requisite {Burgess~~ 1983, 1-einri chs ~ ~ al. (1983) during laboratory infection of oriental 1976, Batra 1973). On the other hand , at artifl­ musta rd by infected insects. It is unlikely that cially high temperature and moisture, successful swe lling was due to hydration of mucilage since ; nvas ion of rapeseed with concom i t ant destruction the underlying material contained structures re­ of cotyledon ce l ls by As pergillus . Penicil li um, sembli ng vegetative yeast cells {Fig. 7). and Verticlllium was accomplishec without prior Seed coat penetration phys i ca 1 damage to the seed (Schws ~ .!!.,_ 1g82 ). Evidence for the spread of yeast infection It is very probable that oriental nustard would be through the seed coat was taken largely from re­ attacked by many fungi in a sim· lar successful sults obtained using fluorescence microscopy tech­ manner under the same abusive storage conditions niques (Fulcher 1982 , Yiu ~.!!.,_1982). without physical injury (Holley ard Ti mbers 1983).

148 ORIENTAL MUSTARD INFECTION BY NEMATOSPORA

Schans et al. (1982) traced the invasion 3. Batra LR. (1973). Nematosporaceae (Hemiascomy­ route and found th"at the inoculated fungi crossed cetl dae): Taxonomy , Pathogenicity, Distribu­ the seed coa t tissue and entered the rapeseed tion and Vector Relations. U.S. Dept. Agri­ cotyledon withOJut apparent diffi culty. Once below culture Tech. Bulletin No. 1469, 71 pp. the pa l i sade cells of the seed coat, the fungi 4. Burgess L, Dueck J, McKenzie DL. (1983). In­ went l a t eral l y amo ng the crushed parenchyma. In sect vectors of the yeast Nematospora coryl i ou r study of irnfected oriental mustard , some lat­ in mustard, Brassica i uncea , cr ops in south­ era 1 moveme nt (of the yeast hyphae may have taken ern Saskatchewan. Can. Entomologist 115, 25 - place abo ve the aleurone cel ls, but major lateral 30. - growth oc curred below the aleurone layer and al ­ 5. Butler JL , Vanderzant C, Carpenter ZL , Smith most fill ed t he e ntire hyal ine layer. Intra- and GC , Lewis RE , Dutson TR . (1980). Influence extracell ular gro wth of the fungi and yeast in of certain processing steps on attachment of cotyledonous cell s was s imilar in both k inds of microorganisms to pork skin. J. Food Pro­ seed . tect. 43, 699-705. In contrast to results obtained by Heinri chs 6. Carmo- SoUsa L. (1970). Nematospora ~. in: et al. (1976) , ~W h o used soybeans inoculated with The Yeasts. J. Ladder (ed.) , 2nd Edition, NematOspora , or i ental mustard cotyledon tissue was North- Holland Pub l. Co ., Amsterdam , 440 - 447. not deeply penetrated by invading Nematospora . 7. Ch andra S, Narang M, Srivastava RK . (1981). Substant ia l growth by the yeast occurred in the Stud ies on seed microflora of oilseeds in hya 1 i ne 1 ayer b etween the seed coat and embryo. India. Part 1. Qua litati ve and quantitative It i s possibl e that myros ine granules in cotyledon estimations. International Biodeterioration cells may pl ay a role in the natural seed defence Bul l. 17, 71 - 75. system to prevent deep penetration by microor ga n­ 8. Co wlen MS, Mars hall RT. (198<). Soaking of isms into cotyledon tissue. Work on the autotoxi­ mustard seeds to release microorganisms in city of musta r d seed to the yeast Nematospora is maki ng plate counts. J . Food Protect. 11. continuing. 340, 344. 9. Fulcher RG. (1982). Fluorescence microscopy of cereals. Food ~1icrostructure 1, 167- 175. Support was obtained for the hypothesi s that 10 . Heinrichs EA , Lehman PS , Corso IC.(1976). the infection process in the seed was i nitiated by Nematospora .f....Q_Q'_]J_, yeast-spot disease of physical injury. This injury was probably caused soybeans in Brazil. Plant Disease Repo r ter during the feeding activity of contaminated in­ 60 , 508- 509. 11. HoiTey RA , Timbers GE . (1983). Nematospora sects with pierci ng-suck ing mouth parts. Areas of destruction in mustard seed by microwave and apparent phy sica l injury were found adjacent to moisture treatments. Ca n. lnst. Food Sc i. s ites of yeast infection on the surface of the Technol. J • .!.§., 68 - 75 . seed coat and on the cotyledon surface. Vegeta ­ 12 . Leistner L, Neumayr L, Forstmeier G. (1981). tive ye-ast cel l s and hyphae were seen to penetrate through the seed coat and to grow laterally at the Verteilung von Mikroorganismen in und auf hyalin e layer bet ween the seed coat and cotyle­ GewUrzen, am Beispi el Pfeffer. (D i stribu­ dons. All morphological forms of the yeast were tion of microor ganisms in and on spices , found b(lth inter- and intracell ularly with respect as illustrated by pepper). Fleischwirtschaft to the coty I edon ce 11 s. 61 , 630- 632. 13 . Martinez AT , Calvo MA, Ramirez C.(1982). Scan­ Acknow l edgments ning electron microscopy of Penicillium co­ ni dia. Antonie van Leeuwenhoek 48, 235 - 255. Or i ental mustard seeds with a high frequency 14 . Pivn i ck H. (1980). Spices, in: i~icrQbial Ecolo­ of yeast infection were kindly provided by Dr. L. gy of Foods. Vol. I I. J . M. Sill iker and R. P. Burgess (Ag riculture Canada, Saskatoon) . Techni­ Elliott (eds. ) , Acad . Press, New York , 731 - cal ass.istance provided by Mr. G. E. Mi llard and 751. Mrs. Paul a A11 an- Wojtas is gratefully acknow 1edg ­ 15. Scha ns J , Mills JT, van Caeseele L. (1g82). ed. The authors wi sh to thank Drs. M. Ka 1 ab and R. Fluorescence microscopy of rapeseeds invaded G. Fulcltl er (Agricu l ture Ca nada , Ottawa} for their by fungi . Phytopathology Jl, 1582- 1586. advice and assistance throughout this study. The 16. Spec k ML . (ed.) , (1976). Compe ndium of Methods Electron Microscope Cen tre, Research Branch , Agri ­ for the Microbial Examinat i on of Foods. Am. culture Canada in Ottawa provided faci 1 ities for Publ. Health Assocn., Washington, DC, 226 - the SEfl study. 227. This paper is Contribution 552 from Food Re ­ 17. Vaughan JG , Hemmingway JS, Schofield HJ . search Institute, Agriculture Canada, Ottawa. (1963) . Contribu tions to a study of varia­ tion in Brass i ca iu ncea Coss and Cze rn. J. Linn. So~58 , 435- 447 . 18. Vaughan JG, PhelanJR , Denfo r d KE. (1 g76). I. Aoba R. (1972) . Histological observati ons of Seed studies in the Cruciferae, in: The seed coat in Brassica juncea Coss. Japan J . Biol ogy and Chem istry of the Cruciferae. Breed. 22, 323-328. J .G. Vaughan, A.J. Macleod, and B.M . G. Jones 2. Ay r es JC;Mu ndt JO, Sa nd ine WE . (1980). Spi ces (eds. ). Acad . Press, New York, llg- 144. and condiments, in: Microbio l ogy of Foods . 19 . Virtanen AI . (1962). Some organic sulfur com­ W.. H. Freeman and Co. , San Francisco, 249- pounds in vege t ables and fodder plants and 260. their significance in hu man nutrition. An ­ gew. Chern . I nternat. .!.. zg9- 308.

149 R . A. HOLLEY, B . E . PHIPPS - TODD and S.H . YIU

20. Vo n Hofs ten A. (1g74). The ultrastructure of Authors: Undoubtedly this is true. Although less seeds of some Brassica species - new sources c learly resolved, irregular surfaces of vegetative of seed prot ein. Svensk Bot ani sk Tidskri ft. yeast cel ls are al so visible in the seed lesion 68. 153-163. viewed by SEM in Fig. 8. 21. YiU, SH, Peo n H, Fulcher RG, Al tosaar I. (1982). The mi croscopic structure and chem­ Reviewer V: Is a reference s train of the infect- istry of rapeseed and its products. Food ing organism available? Microstructure .!.. 135-143. Authors: Yes, the Nematospora culture studied has ~osited in the co llection of the Centraal Bureau voor Schl mmel cultures, Baarn, the Nether- 1 ands and has been ass i gned CBS#8199. The cu lture 0. N. Holcomb: Cou ld you give more detail of th e is also preserved at the National Mycological 1Tu0rescence microscopy technology and provide Herbarium, Ottawa, Ontario, Canada K1A OC6, where some warnings as to artefacts with thi s technique? it is given the number DAOM 187446. Authors: The formation and recognition of arte­ fa c t s are important aspects of this and other tec hniques i n microscopy. Substantial additional information on the applications and limitations of fluorescence microscopy are provided in the paper by Ful cher (1g82) cited in the bibl i ography.

L. van Caeseele: In view of the differe ntial color ODtaTn~ans et al. {1982) using Ac ridine Or ange and MalachiteGreen, did you try comb ina­ tions of s tains such as this? Authors: The major part of our work was done ~e latter was published and thus the dyes mentioned were not used. In view of the success ac hi eved by Schans et al. (1982) with rapeseed, they may be quite apprDprTate for use with mustard as well. Fig. 12. Aniline Blue- stained cultu r e smear show­ ing yeast cells and spores (arrow). Photographed L. van Caeseele: Fig. 6 shows puncture marks (ar­ using FC I . ToWST:" ~scussion you speculate that these may be caused by the false ch inc h bug. If this Fig. 13. Congo Red- stained GMA-embedded paradermal were so, would you expect smooth edges on the section of infected mustard seed showing the epi­ pun c ture hole? Would the_ holes vary in diameter? dermal mucilage (M) and the yeast (Y) structures. Is the diameter of the false ch inch bug proboscis Photographed using FC III. known? Authors: Insects , which could be respons ibl e for Fig. 14. Congo Red-stained GMA - embedded paradermal inflic ting pun cture damage upon these c rops , vary section of the palisade layer (P) of infected in s ize and thus the lesions they cause also vary mustard seed penetrated by yeasts (Y) in an area in their dimensions. Indeed,the male false chinch of seed tissue showing signs of disorganization. Photographed using FC II. bug is significantly smaller than the femal e. The proboscis of the false chinch bug (the most like­ Fig. 15. Aniline Blue- Calcofluor White- stained ly insec t to be involved) female measures approxi­ GMA section of infected mustard seed coat showing mately 50-80 lJm in diameter. Th i s inc ludes an epiderm is (E) , palisade (P), pigment (arrow) , outer sheath which does not penetrate. Inside the aleurone (A), and hyaline (J/) layers. sheath are 4 stilets, two of which c ut the hole. Photographed using FC I . The diameters of the "holes" in Fig. 6 are within the size range of those which would be produced by Fig. 16. Congo Red- stained GMA section of infected these insects {10-20 11m). The edge of these punc­ mustard seed showing yeast spores (arrows) spread­ ture wounds would initially be ragged, but as the ing throughout the hyaline (H), palisdde (P), and seed matured and dried, one would expect changes aleuron e (A) layers. to occur in the perimeter of these l esions. Photogr aphed using FC II. J. G. Vaughan: Are the authors interested in car­ Fig. 17. Congo Red- stained GMA section of infected r:Ylng out a cantrall ed experiment on yeast with healthy B. juncea seed? mustard seed showing yeast cells (Y) at the peri­ phery of the damaged cotyledonous cells (* ). Authors:Yes:-ana we would be especially interest­ Photographed using f'C II. ecrrns-tudying the progress of yeast infec tion during seed maturation. It is an interesting con­ Fig. 18. Congo Red- stained GMA section of infected tradiction that the host seed is quite to xic to­ mustard seed sh:Jwing yeast spores (arrows) inside ward the yeast parasite. an infe cted cotyledonous cell ( * ). Photographed using FC III. ~ .!i.!_ Humphreys: Cou ld the folds shown in the vegetat1ve yeast cells (Fig. 12) be artef acts of Errata : The correct marker on Fig . 18 is 20 lJm . drying?

I 50 ORIENTAL MUSTARD INFECTION BY NEMATOSPORA

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