View of Annual variations in the microflora of some varieties of Finnish malting barley

V01.4:407^t18.

Annual variations in ^the microflora of some varieties of Finnish malting barley

TapaniTuomiandHeikkiRosenqvist

HelsinkiUniversityofTechnology, Departmentof^ChemicalEngineering, LaboratoryofBiochemistry andMicrobiology, FIN-02150 Espoo, Finland.

Three majorFinnish malting barley varieties werestudied for annualvariations inthe incidence of seed-derivedfungi,bacteria and actinomycetes. In 1990-1992, 114characterizedfungal, 59unchar- acterized bacterial and 12uncharacterized actinomycetal isolates ^were extracted from samples of

seed intended foruse ^inmalting.

When^the yieldof^the plant hormone, indole-3-aceticacid^(lAA), from enriched microbial cultures was weighed against the microbial biomass and the endogenous lAAconcentration of the barley harvests,itwasconcluded thatpotential exists for bacterial lAAproduction in biologically signifi- cant amounts,givensomeminor annual variations.

Asexpectedfrom the average rainfall and temperatureduringthe growing season, microbial counts inallcultivarswerehighest in 1992.Most of thefungal speciesfoundwereofsaphrophyticcharac- ter, and field fungiweredominant in the samples. ^Onthewhole,microbial counts and spectrainall samplesconfirmed that each harvest of all cultivarswas ofgood vigourand well suited formalting purposes. Strains ofplant pathogenic character included speciesofSeptorianodorum(Berk) Berk, Drechlera^teres(Sacc)Subraim^& Jain, D.sorokiniana(Sacc) Subram^& Jain andD. graminea (Rab.) Shoem.

Aconsistent difference wasnoted in the microbial infection severities of the cultivars.

Keywords: Hordeum vulgare,plant-microbe interactions,indole-3-acetic acid Abbreviations: cfu,colony-forming units; lAA,indole-3-acetic acid

ntroduction

A wide range ofbacteria, yeastsand moulds col- onize the surface andouterlayers ofmaturebar- ley kernels both in the field and during storage (Briggs 1978, Flannigan 1974).Germination in moist soil exposes the graintoanadditionalspec-

trumof microorganisms under conditions favour- able for microbial growth. Penetration of micro- organisms is usually arrested by cuticularized layers and highly lignified walls of the surface layers of the grain. Microbial growth appearsto extendto the testa-nucellarcuticle, which acts as abarriertofurther penetration.However,these mechanical barriers to microbial invasion may

©Agricultural ^Science inFinland Manuscriptreceived January 1995

be weakened when grains crack during drying or harvestingor when the surface layers soften at the high moisture contentsrequired for ger- mination (Briggs 1978). Microflora associated with the graincan cause gushing of undesirable flavours in beer and produce harmful_mycotox- ins (Amaha and Kitabatake 1981, Chelkowski

1991). These microbes influence the enzymatic activity of wetted grain and alter the quality of malted grain, andonseveral occasions microbi- al activity has been notedto interfere with ger- mination (Briggs and McGuinness 1992).

Much remainstobe learnt about the mecha- nisms of microbial actiononcereals. In normal circumstances, microbial production ofenzymes suchasP-glucan solubilase(Yinetal. 1989)and cellulases (Hoy etal. 1981) is of importance,as arereduced oxygen _contents due _to microbial activity(Doranand Briggs 1993).One factor that tends ^tohave been overlooked is the microbial production of the plant hormones that influence the germination and growth of the grain. In plants, absolute and relative levels of hormones areinstrumental in controlling phenomena such asgrowth, differentiation, structural organisation and passage from the vegetativetothe reproduc- tive phase,as well as other developmental and dynamicevents. The effect of endogenous hor- monegradientsonplantscanoften be mimicked by applying hormonestoplants externally, and thus also by microbes producing and excreting hormones(Davies 1990, Gogola 1991, Haahtela etal. ^1990,Loper and Schroth ^1986,Rademacher 1992,Rademacher and Graebe 1979, Serradaet al. 1982).

Microbe-derivedplanthormones ofpotential interest for the development, germination and growth of barley kernels include compounds from all main classes of planthormones,but only bacterial indole-3-acetic acid (lAA) has been estimatedtooccurinamountsof any significance (Tuomi ^etal, 1994,1995). In screening barley grain for microbial lAA production, _we previ- ously used three Finnish malting grade barley cultivars of the 1990 harvest. As would beex- pected, the microbial population of barley var- ies not only from cultivar to cultivar, but also

from year to year. In ^an effortto see how our results hold up^to annual variations in the mi- crobialflora, we in thepresent study examined variations in the incidence ofbacteria, and also of fungi and actinomycetes, in thesame three malting barley cultivars in 1990, 1991 and 1992.

Our main objective was toevaluate the annual variability in microbial plant hormone produc- tion in barley, but in doing so we concurrently acquired additional informationonthe variations in the microbialspectraandcounts of barley.

Previous investigations have givenus agood picture of the_spectraof epiphytic mouldson the roots, stembases and leaves of Finnish barley (Mäkelä 1972,1977a,b, Mäkelä and Mäki 1980, Mäkelä and Parikka 1980).Less isknown, how- ever, about the seed-borne fungi of malting bar- ley, and the few studies that have been published onthis subject have usedadifferent methodolo- gy and differentbarley varieties fromus(Haikara etal. 1977, Ylimäki 1970, 1981). As a result, several species ^not heretofore reported in Finn- ish barley seedsareintroducedhere, whileatthe sametime many previously encountered species were ^notfound. Sinceourfindings complement the overall picture of the mycoflora of Finnish barley grain,wefocusonthe general microbiol- ogy of thebarley samples, which is of interest

toall those involved with the processing and cul- tivation of Finnish barley varieties.

Barley plants grown intemperate, humidre- gionsareparasitized by about teneconomically harmful bacterial and fungal foliar pathogens, someof which also attack partsother than foli- age.Except forrustsand powdery mildew,most of the foliar-attacking parasites, aswellasmany of the pathogens causing diseases inroots, are seedborne, and kernel infections frequently dis- colour kernels and^causeloss of quality and crop

value (Kiesling 1985, Mathre ^1982).All barley foliar pathogens with the exception ofrustshave associations with infected crop refuse from pre- vious barley crops. The importance of infected

seed in the disease cycle is inversely proportional to the ^amount of inoculum produced from in- fected crop refuse. When cultural practices in- volving little or no tillage and monocultureare

Vol.4: 407^18.

combined,thenewcrop may haveextremely high inoculum potential. Under favourable climatic conditions this maycause severeearly outbreaks of foliar diseaseson plants in the first of the four leaf_stages(Kiesling 1985).With this in mind^- and since the data_were at hand^- _wealso look briefly atthe pathogenicity of the isolated spe- cies, and on the incidence of plant pathogenic species _amongst the seeds of different cultivars from harvest toharvest.

Material and methods

Barley samples

Dry (moisturecontentca. 8wt^%), making-grade grains of the cultivars Kymppi, Kustaa and Hjan Pokkowereused. These cultivarsareamong the four most important barley varieties used by Finnish malt producers (cv. Kilta being the fourth). Kymppi is a two-rowed variety that is also used asfodder (since 1987).Kustaa (mar- keted in 1980)is reasonably good in yield and has a shorter growing season than other two- rowed barley varieties cultivated in Finland.

Hjan Pokko (introduced in 1980) is _a multi- rowed variety that gives high yields and is well

suited^tothe production of high-enzyme-content malt (Lampinen 1989).

Fungi and bacteria were isolated from all three cultivars of the 1990, 1991 and 1992 har- vests.The samples were taken directly from a malting plant as soon as the new harvests be- came available. Microbes were isolated within oneyear ofharvesting in 1991-1993. Five par- allel plateswere prepared ^ateach isolation and enumerationstep.

Isolation and enumeration of fungi and bacteria

Fungi, other than Fusaria^,and bacteriawereiso- lated from spread plates prepared from milled

(Frithsch Pulverisette 14, sieve size 0.5 mm) grains suspended in physiological salt solution.

Potato dextrose agar(PDA, Biokar Diagnostics) containing 0.5 g/litre ampicillin wasused for iso- lating the fungi, and Plate-count agar (Merck) with 0.01 g/litre cycloheximide for isolating the bacteria. Fungiwereinoculated in both daylight and under near-UV radiation from ablack light (Philips, TLD 36W/08) with a 12 h light-dark cycle toinduce sporulation. To distinguish iso- lates, fungiweregrownon Maltextract(Merck), Czapek-Dox ^(Oxoid) and Wort(Difco) agars, as well as on PDA, in both darkness and light.

Viable^countsoffungi and bacteria in grainswere calculated from dilution plates.

Fusarium spp. failed to show up on spread plates andweretherefore isolated by direct plat- ing of kernels on Modified Czapek-Dox agar containing iprodione and dichloran (CZID me- dia) according to the method described by Abildgren etal. (1987). Plated kernels were in- cubated under a daylight lamp (Sylvania, 36W Activa 172). Fungal isolates were identified at the Centraalbureau voor Schimmelcultures in Baarn, The Netherlands. Isolates belonging to the _samespecies _weretreated separately if they had been isolated from different cultivars. The percentage ofkernels contaminated with isolates served as a quantitative measure of Fusarium contamination.

Actinomyceteswere isolated and^enumerat- edon spread plates prepared on Starch-casein agar(10 g/l soluble^starch;0.3 g/1^casein; 2.0 g/I KNO_s^; 2.0 g/l ^NaCI; 2.0 g/l K,HP0₄; 0.05 g/l MgSQ₄x 7H₂^0; 0.02 g/l CaCO,; ^{0.01 g/l} FeS0₄x7H,0; 15.0 g/l Bacto agar,^Difco), con- taining 0,0125 mg/ml cycloheximide, as de- scribed by Randetal. (1976).

Analysis of lAA

The lAA content of barley grainwas analysed according^tothe method described by Tuomi and Rosenqvist(1995). The method involves purifi- cation by solvent partitioning and thin layer chro- matography, preparation of trimethylsilyl deri-

Table 1.^Variationsinaverage rainfall and temperatures(T) inthe 1990-1992growingseasons.Values have been summedovercultivationregions I andIIas^coveredbyMustonen et al. 1994,and summed values dividedbythe number of observationpointsto get average values forcombined cultivationregions Iand11.

Data from The Finnish Meteorological Institute.

Period 1990 1991 1992

Rainfall T Rainfall T Rainfall T

(mm) (C°) ^{(mm) (C°) (mm) (C°)}

01.05- 9 9 12 7 4 11

31.05- 5 15 12 12 7 16

30.06- 16 16 17 17 26 16

30.07- 20 16 30 17 31 14

30.07- 13 17 16 20 14 16

09.08- 31 17 58 15 39 15

19.08- 16 13 16 16 39 12

01.05-28.08 218“ 107^h 253* 99^b 205“ 110^b

aTotal rainfallduring period

b Deviation of the termicperiod ofgrowthfrom reference value (100%).

vates with /V,O-his(trimethyl-silyl)trifluoro- acetamide (BSTFA) and analysis by gas chro- matography-mass spectrometry^(GC-MS),using selected-ion monitoring (SIM).

Results and discussion

lAA production ^{in bacteria}

Nine^outof^tenbacteria(88%)in cultivars Kus- taa,Kymppi and Hjan Pokko of the 1990 har- vest were previously shown to produce lAA (Tuomi etal. 1994, 1995).To estimate the phys- iological significance of the presence of lAA- producingbacteria,lAA production per bacteri- al cell in the liquid growth media of the cultured bacteriawasweighed against the bacterialcounts and the endogenous lAA concentration of bar- ley grains. On the basis of thesecalculations, we estimated the bacteria in steeped barley to be capable of lAA production in ^amounts of bio- logical significance (Tuomi et al. 1994, 1995).

Bacterial counts in the 1991 and 1992 harvests furthersupportthis assumption, since thecounts in all sampleswerehigher than the lowest ones found in the 1990 harvests (Fig. ^1).As_{a conse-}

quence, bacterial lAA production would fall within the limits estimated for the crop of 1990.

To further clarify this point, the endogenous lAA concentration in dry kernels ^(cv. Kymppi, 1992 harvest)_was analysed. The result, 123^± 63 g/g freshwt,corresponds well with the lAAconcen- trationonwhich the 1990 estimateswerebased (195⁺99 g/g fresh^wt).Counts of fungi,on the otherhand, weretoo low in both 1991 and 1992

- asthey werein 1990(Tuomi etal. 1994,1995)

- for fungal lAA production tobe a factor of significance.

Total counts of fungi ^and bacteria

In 1992, Augustwas inclinedtobe adamp and relatively cold month(Table 1).Just before har- vesting then,^atavery crucial timein the devel- opmentof the grain microflora, conditionswere wet, and the watertaken up by the seeds could

not evaporate.As expected, the highest bacteri- al and fungal counts in all cultivars werethere- fore found in 1992 (Fig. ^1), when the weather favoured the attachment and proliferation of these organisms. In 1990 and 1991 no clear dif- ferences were seen in the profiles of the total

counts of fungi and bacteria.This, too, was to

Vol.4: 407—418.

be expected since therewere no marked differ- ences in either average temperatures orrainfall between these years.

In all, 59 uncharacterized bacterial isolates were extracted. Eighteen of these isolates were from the crop of 1990⁽⁶ in cv. Kymppi, 7 in Kustaa and 5 in Hjan ^Pokko), 24 from that of 1991 (8 in _cv. Kymppi, 6 in Kustaa and 10 in HjanPokko)and 17 from that of 1992⁽⁶ incvs.

Kymppi and Kustaa and 5 in Hjan Pokko). As seen in Figure 1,_cv.Kymppi wasclearly richest andcv. Hjan Pokkopoorestin both bacteria and fungi. Thereasonsfor thisare^notclear,although one may be thatcv.Hjan Pokkohas,interms of surfacearea, the largest kernels and consequently less surface of attachment per grain than culti- varsKymppi and Kustaa.

Viable^counts of actinomycetes ^{in all sam-} pleswere low in comparison with both bacterial and fungal counts^(< 10²cfu/g fresh_wtof grain).

Noattempt was made^tocharacterize these spe- cies.

Fungal spectra

A confusing array offungi, 114 isolates ofyeasts and moulds belonging ^to48 different species, wereextracted from the three cultivars in 1990- 92 (Table 2).The commonest classes arelisted in Figure 2, which shows thatmostof these spe- cieswere^present in all three cultivars and some of them in all three harvests of the cultivars.

Xerophilic storage fungi, namely Aspergil- lus and Penicillium spp.,werein_aminority in all samples (Fig.2)eventhough the isolation meth- od whould have favoured the appearance of these sporulating species. The levels of both Aspergil- lus and Penicillium spp. appeartorise, howev- er,when barley is steeped (Douglas and Flanni-

gan 1988, Haikara etal. 1977)and it is there- fore importantto notethat theywere present in

mostof the samples (Fig. 2). As wellasharmful mycotoxins, members of both Aspergillus and Penicillium generaareknown to produce vari-

ous plant hormones (Pegg 1984, Tuomi et al.

1994, 1995). Aspergillus spp. and Penicillium spp. also produce many spores thataredissemi- nated by air and ^{can cause}respiratory diseases in people and animals (Mathre ^1982).Counts of these organisms in the grains would have ^tobe higher than those reported here, however,before they would give any causeforconcern. On the contrary,the relatively lowcountsof these harm- fulstoragefungi indicate that the grainswereof good vigour and well suited for malting. One likely reason for the relatively low percentage ofstoragefungi is that the sampleswereall tak- enwithinayear of harvesting, and the mycoflo- ra would still be dominated by field fungi (Briggs

1978,Burger and Laßerge 1985).

The mostconspicuous field fungi were spe- cies ofRhodotorula, Cryptococcus and Aureo- basidium(A. pullulans), all yeastlike organisms of widespread occurrence (Jay 1986),and spe- cies of Drechslera^, Cladosporium and Mycelia sterilia (Figs ^2-3).Drechslera spp. include spe- cies ofplant pathogenic interest(see below)and even though not normally cited as occurring in high numbers in barley (Briggs 1978, Douglas and Flannigan 1988, Flannigan 1974, Flannigan Fig. 1.Total annual viable counts(cfu/gfresh wt of grain)

offungiand bacteriaincvs.Kymppi, Kustaa andHjanPok- koin 1990-1992.

Table2. Fungal speciesisolated fromcvs.^Kymppi,Kustaa and Hjan Pokko inharvests of1990, 1991and 1992.

Strain Number of isolates

cv.Kymppi cv.Kustaa ^cv.HjanPokko

’9O ’9l ’92 ’9O ’9l ’92 ’9O ’9l ’92

Acrodontium crateriforme(v. Beyma) de Hoog ^{- - - - - - -} 1

Altemaria altemata (Fr.:Fr.) Keissler 3-1 ^{___ - -} 2

Altemariainfectoria^{Simmons 1 - - - - - 1-1}

Aspergillusflavus^{Link:Fr. 1 2 - - - 1 - 1}

Aureobasidiumpullulans^(deBary)Amaud 1 2 ^{- -} 2 2 ^- I 2

Botrytiscinerea Pers.:Fr. ^{- - - - -} 1

Candida spp. 1 ^{- -}

Chalaraaustiaca (Faut.^&Lamb.)Nag Raj^&Kendrick ^- 1

Cladosporium cladosporioides(Fres.) de Vries 1-1 ^- -1 ^- II

Cladosporiumherbarum (Pers.:Fr.)Link ^{- -} 1

Cryptococcusalbidus (Saito) Skinner^var.albidus ^{- -} 1 ^{- -} 1 1-1

Cryptococcusinfirmo-miniatus(okuniki) Phaff^&Fell ^{- - - - - - -} 1

Cryptococcuslaurentii (Kufferath) Skinner ^{- -} 1 ^{- -} 1

Drechsleraanam.ofPyrenophora graminea ^- 2 1

Drechslera cf. sorokiniana ^{- - - I__ ___}

Drechslera sorokiniana (Sacc) Subram^& Jain ^- 1 ^- 2-3 ^{- -} 1

Drechslera teres (Sacc.) Shoemaker ^- 1

EpicoccumnigrumLink 1-1 ^{- - - - -} 2

Fusarium spp. 5 n.i n.i 1 n.i n.i ^- n.i n.i

Heterobasidiumannosum(Fr.:Fr.) Bref ^{- - - -} 1

Hypocrea pulvinataFuckel,isol. from Piptoporus betulinus ^{- - - -} 1

Myceliasterilia 42 ^- 221 ^- -2

Penicillium aurantiogriseum ^{___ - - - __l}

Penicillium cf.commune I 1 ^- I ^{- -}

PenicilliumcorylophilumDierckx 11

Penicillium crysogenum Thom ^{- - -} 2 I ^{- -} 1

Pichia anomala (Hansen) Kurtzman ^{- - - - - - -} 1

Rhodolorulaglutinis(Fres) Harrison 1 ^{- - - -} I ^- 1 1

Rhodotorulamucilaginosa (Jorgensen)Harrison ^{- -} 1 ^{- - - -} 1

Septorianodorum (Berk) Berk ^{- - -} 1-1

Sordariafimicola(Rob.) Ces.^&De not. ^{- - - - - - -} I

SporobolomycesroseusKluyver ^&vanNiel. ^- 1 ^- II

Trichoderma viride Pers:Fr. 1

UcladiumbotrytisPreuss ^{___ ___ __l}

Unidentified spp. 2-2 ^{- -} 5 1

Williopsiscalifornica(Lodder) Krasil’nikov ^{- - -} 1

"'notisolated

and Healy 1983, Haikaraetal. 1977),these fun- gi appeartobe of special importance and spread in Finnish barleys (Mäkelä 1972). The other groups^mentioned, A. pullulans and Cladospo- rium spp. in particular, are more frequent ac- quaintances among the mycoflora of healthy

barley grain (Briggs 1978, Douglas and Flanni- gan 1988,Flannigan 1974,Flannigan and Healy

1983. Haikara ^et al. 1977). The Cladosporium spp, found were either isolates of C. cladospo- rioides Fres. de Vries_orC. herbarum(Pers.iFr.) Link. C. herbarum (Pers.:Fr.) Link, which was

Vol.4:407-^lB.

Fig. 2.Viable counts of the fourteen mostprominent fungalgroupsincvs.Kymppi,Kustaa andHjanPokkoin1990-1992,

found only incv.Kymppi of the 1992harvest,is asaprophyte that frequently parasites weakened or damp grain but, much like C. cladosporio- ides Fres. deVries, is relatively unimportantas a seedborne infectant (Flannigan and Healy

1983).

Many of the fungal groups listed in Table 2 have previously been found in barley varieties grownin Finland. Haikara etal. (1977)isolated fungi from four Finnish barley varieties(notcv.

Kymppi, Kustaa orHjan Pokko), and among the sevenmostcommonfungal genera listed by them all but Cephalosporiumare included in Figures 2 and 3. Clearly, however, most of the fungal groups listedin Figures 2 and 3are notspecific for Finnish barley, ^most having been citedas commonfungi in malting barley by workersus- ing both direct plating and spread plating in dif- ferent countries the world over(Briggs 1978, Douglas and Flannigan 1988, Flannigan 1974, Flannigan and Healy 1983, Haikara et al.

1977).Ylimäki(1970, 1981)has also investigat- ed the mycoflora of several Finnish barley vari- eties (notcv. Kymppi, Kustaa or Hjan Pokko), buteventhough his studies spanned several years and covered grain fresh from harvest aswellas

samples of stored grain, he does ^not mention many of the species listed in Table 2. He did, however,list even morespecies of various gen- era thanaregiven in Table2. Thereasonfor this spread in the results is probably that these pre- vious studies utilized direct plating of intact ker- nels on solid media. Direct plating of fungi on agarorfilter paper is anefficient way of isolat- ing filamentous fungi (moulds) from intact seeds but, in our experience, is less suitable for _ex- tracting bacteria and yeast-like organisms, which tend_tobe overgrown and displaced by moulds.

Hence,the number ofyeaststhat showedon our mediawas higher than would be expected from direct plating. Our results are altogether _more compatible with thoseof, for _instance,Flanni- gan (1974), who used spread plating aswellas direct plating to investigate the fungal flora of Bulgarian barley.

In all samples, contamination with Fusari- um spp.wastoo rarefor themtoappearon dilu- tion plates. Direct plating ofcornsfrom the 1990 crops gavea maximum of 10% of grains con- taminated with Fusaria (in cv.Kymppi) and a minimum of0% (in cv.Hjan Pokko) (Tuomiet al. 1995).

Fig. 3.Mean values of viable countsof the fourteen mostprominent fungalgroupsincvs.Kymppi,Kustaa and HjanPokko in 1990-1992,expressedaspercentages of total cfus.

Vol. 4: 407^18.

Incidence of plant pathogenic fungi

All samples contained onlysmut-free,apparently healthy grain. Noreports of outbreaks of major epidemics of economic importance that would concernthe harvests of the three cultivars stud- ied here have _cometo ourattention. Neverthe- less, some species of plant pathogenic impor- tancewerefound whose incidence is difficult_to linktoharvesting yields (Mustonen^etal. 1994).

Septoria nodorum(Berk) Berk was present in _cv.Kustaa of the 1990 and 1992 harvests. S.

nodorum,citedas moderatelycommon on two- rowed barley in Finland (Mäkelä and Mäki

1980),causes darkening ofstems and leafspots aswellasfoot androot rot(Mäkelä and Parikka 1980). Seeds are aknown sourceof primary in- oculum for this fungus andcanharbour S. nodo- rumfor uptothree years.

Other plant pathogenic species found include species ofDrechslera, which occurred in all three cultivars and sometimes in more thanone crop (Table 2, Fig. 2);they were,in fact, among the

ten mostabundant fungi found (Figs ^2-3).

The 1991 crop of cv. Kymppi contained Drechslera teres (Sacc) Subraim ^& Jain (an- amorph of Pyrenophorateres),the causal organ- ism ofnet blotch (Kiesling ^1985).This fungus persists fromone growing seasontothe nextas seedborne mycelium or as pseudothecia in in- fested host residue. Seedborne mycelium prob- ablyservesto introduce the pathogen into fields previously free ofnetblotch (Mathre 1982). In Finland, the fungus has been observed tocause leafspot onbarley, but is probably of minoreco- nomic significance (Mäkelä 1972).

Drechslera sorokiniana(Sacc) Subram ^&

Jain (synonym of Helminthosporium sativum and Bipolaris sorokiniana, anamorph of Cochliobo- lussativus)was found in all three cultivars and all three harvests (Table 2, Fig. ^2).This fungus is, together with Fusarium culmorum and F.

graminearum, citedas a cause ofrootrot and seedling blight in barley (Mathre 1982). Acom- mon fungus in Finnish barley seed, it may have economic significance, as yield losses of up to 11%, witha meanof 6.3%, have been reported

in field experiments (Kurppa 1984,

1985

Seedborne inoculum is themostsevereform of primary inoculum and may result in dead or stunted seedlings (Kiesling 1985, Mathre 1982).

The infection incidence of Finnish barleys (cvs.

Kymppi, Kustaa and Hjan Pokkonot considered) and invasion of D. somkiniana to the internal cell layers of kernels is known ^tovary consider- ably, and not all varieties are susceptible to pathogenesis (Kurppa

1985

losses have been reported (Mustonenetal. 1994), it is possible thatnoneof these three cultivars is susceptibleto D. sorokiniana.

The causal organism of barley stripe, Drechslera graminea (Rab.) Shoem. (anamorph ofPyrenophora ^graminea),wasisolated from the 1991 and 1992 crops ofcv.Kymppi. This seed- borne disease has beencommonin Finland fora long time and continuestobe so(Mäkelä 1972, Mäkelä and Mäki 1980).The primarysource of inoculum of this disease, too, is mycelium in infected seed producing systematically infected plants (Teviotdale and Hall 1976).Infected plants produce fewseeds,and those thatareformedare shrivelled. Losses aretherefore directly propor- tional to thepercentage of infected plants ina field. A number of spring barleys have beenre- portedasresistant^to thedisease, whereas most

winter barleys are susceptible (Mathre 1982).

Like all malting barleys cultivated in Finland, cv.Kymppi, from which isolates of P graminea were found,is aspring barley.

Conclusion

In addition^tothe action of the above plant path- ogenic fungi, fungi of the generaPenicillium, Aspergillus,Fusarium, and Alternaria_areknown

tohave deleterious effectsonthe quality ofFinn- ish barleys owing to the production of various toxins. For these species tobe of significance, though, higher counts than those reported here (Fig. 2) arerequired (Hietaniemi and Kumpu- lainen 1993). In damp years, the infection of Finnish barleys with Fusarium species in par- ticular has caused problems in the processing of

the grain (Haikara 1983). The smallpercentage of grains contaminated withFusaria inour sam- ples does not, however, give cause forconcern and, on thewhole,all samplesseemtohave been drawn from barley that^- atleast intermsof fun- gal and bacterial counts ^- were well suited for malting.

Depending onthe^amounts released, the role of the bacterial lAA reported here might be to inhibitorstimulate germination while also stim- ulating enzymatic activity (Doran and Briggs

1993, Kieninger 1983, Kieninger and Blohm

1983, Li and Rehmanji 1991,^{Yamada 1984).In} malting trials, similar amounts of added lAA have been noted toenhance the effect of added GA₃ (Kieninger 1983, Kieninger and Blohm 1983, Palmer 1971).Whatever itsrole,bacterial lAA seemstobe anintrinsicpart of the interac- tion between germinating barley grain and its hosting bacterial flora.

Acknowledgements.The authors thank MSc. Jouni Ilvesoksa for technical assistance. The workwasfinanciallysupport- edbythe Foundation for Biotechnical and Industrial Fer- mentationResearch,Finland.

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SELOSTUS

Vuosittaisia vaihteluja suomalaisten mallasohrien pieneliöstössä ja niiden vaikutus liotetun ohran indoli-3-etikkahappopitoisuuteen

TapaniTuomijaHeikkiRosenqvist

Teknillinen korkeakoulu

Kolmenyleisimmänsuomalaisenmallasohralajikkeen jyvien bakteeri-, mikrosieni- (hiiva ja ^home)ja sä- desienipopulaatioiden vuosittaisia vaihteluita tutkit- tiin vuosina 1990-1992. Työnaikana mallastukseen käytetyistä ohraeristä eristettiin yhteensä 114karak-

terisoituamikrosieni-, 59tunnistamatonta bakteeri ja 12tunnistamatonta sädesieni-isolaattia.

Kasvihormonin, indoli-3-etikkahapon, saantoa jyväperäistenmikrobien rikastetuissa puhdasviljel- missä verrattiin mikrobien biomassaan jyvissä ja jy- vienindoli-3-etikkahappopitoisuuteen. Todettiin, että ottaenvuosittaiset vaihtelut^huomioon, bakteereilla

onkyky tuottaaindoli-3-etikkahappoa biologisesti merkittävissä määrin.

Kuten satokausien aikaistenkeskilämpötilojen ja sadesummien perusteella saattoi odottaa, bakteeri-,

hiiva-jahomemäärät olivat kaikissalajikkeissakor-

keimmillaanvuonna ^{1992.Suurinosa}eristetyistähii-

voistajahomeista lukeutuvatsaprofyytteihin. Domi-

noivialajejaolivatpellolta peräisin olevat Rhodoto- rula, Cryptococcus, Aureobasidium, Drechslera, Cla- dosporium ja Mycelia sterilia. Varastosienet olivat tätenvähemmistönäjakaiken kaikkiaan vaikuttaisi, ettäkunkin kolmen lajikkeenkaikki kolme vuosiker- taakoostuivatmikrobiologisesti hyvälaatuisesta, hy- vinmallastukseen soveltuvasta viljasta. Kasvipato- geeneiksi luettavia mikrosienilajeja olivatSeptoria nodorum(lehti-ja tähkälaikku) sekä Drechlerateres, D.sorokinianaja D. graminea (aiheuttavat mm. juu- rimätääjataimituhoa).

Mikrobi-invaasioiden määränhavaittiin poikke- avanselvästi ja johdonmukaisesti lajikkeesta toiseen.