V{ISCONSIN STATE UNIVERSITY - LA CROSSE
GRALDUATE COLLEGE
Candidate: Frederick L .. Sc.hmude
I recoDlruend acceptance of this ser.ninar paper to the Graduate College in partial fulfillrnent of this candidate l s requireI:D.ents for the degree .Nlaster of Science. The candidate has con1.pleted his oral se:minar report..
~ C.' ~~K£.-4 ,;:> Sem:ina r Paper A.dvi s or
This seminar paper is approved dthe Graduate coO:1 ~b!1-97f) ~iIi~ I ate.- Dean, ura~te College i\N INVESTIGA,TION OF AQ lLL\ TIC
00lvlYCETES IN FIVE AREAS
OF PLEASAN'.T' PR~4..IRIE TO\VNSHIP
by
Frederick L. Sc}snude
ABSTRA.CT
The purpose of this study \vas to deterrnine the types of
OODl.ycetes present in selected aquatic habitats in Pleasant Prairie
Tovvnship, Eenosha County, \Visconsin. Pleasant Prairie V\lCtS se lee-ted because no previous studies of this nature had been made
The five habYitats chosen \vere the Des Plaines River, Lake l\.1ichigan, Barnes Creek, 1\.1ahoney I ereek and lv1ahoney I s Pond..
The ITlethod of obtaining the sample s Vile 1'e a lTIodificatioD of the trapping techniqu.es proposed by Patterson in 1967 and Nelson. in
1969. Baby food jars containing either hell'll? seeds or dead fhes
\ve 1'e placed in a styrofoam float and covered \vith chee se cloth. we r e then pa rtially fi11ed v;,rith wate rand inve.rted n1.outh down in the su1'.face \;vater of the selected site.
Through the use of the traps and pu.re culture techniques on Di.fco Corn Meal agar, it \vas determined that there \vere at least five types of Oomycetes present. The organisrns found ·were
Saprolegnia ferax (Gr1.lithuison, Thuret), Saprolegnia dic!ina
(Humphrey), i\.podachyella con1.Eleta (1-Iwuphey, Indoh), Dictyucbus
sp., and Pythiurn. Spa
Selected n1.icrographs have been inc.luded to illustrate the
structures involved in classifying these organisms. j\N INVES TIG}\.TION OF AQUA TIC
OOlvlYCE TES IN FIVE i\REAS OF
PLEASANT PRAIRIE TOWNSHIP
A Sen:1inar Report
Presented to
The Graduate College vVisconsin State University, .La Crosse
In Partial Fu.l£illnl.ent
of the Requiren"lents for the Degree
M.a ste r of Science in Biology
by
Frederick L" SchJ:llude
July, 1970 TABLE OF CONTENTS
Pi~.GE
~ ~ l.nt r" 0<1ue ti ()Il $ '" 0 0 • & X Literattlre Revie\"'\l " " ~ " .. " Q .. ., " ., .. " .. ., " ., ., .. 2 Methoel sand 1viat e ria1s ...."""...... ".."...... <> ...... " " ...... , " " " .. " ...... 13 28 ~ ~ ~ ~ ~ ~ Discussi()n . .qs 0 • " " « • 0. .. Q' ., (J: 0 •• t> .. 0. 00 •• .0 .. 9 3 00 u ... 0" (l. (J o;r. Q'1o .... l:: $ " '0 43 TABLE O.F' CONTENTS PAGE ~ ~ ~ ~ ~ Int T odu c ti() n .. 9 Jt g • • A .. • (. • 'tS 0. .. 0 .0 0 ... .. 00 ... .. '" • ... tl 1& • 9 0 eo 0. 0 .:) .. eo 'I:) & "0 'I:) * .. Ot • I Litera.ture Revievv .. " ••.. ,. ., " •• l;> ., "...... Z Meth.ods and 1.1aterials .""." " " " •• "" " .. "' .. " .••• ,... 13 ~ Res"lIltSOlO' lit (r '" ..... '0 " 0' • " • i) Q 0 • 8 Gt • 4) 0 0 • 01 0; 'IiIIo 41 0 Q II 0 • J;) ••• a ••• 01 '0 « Cot • Jt (c • .. 28 I)iscussion " " " .. " .. " .. " "" "" .. " " .. " .. " .. "...... • 32 ~ ~ S ll.ITlITla r y Ol • IQ • .. t,) • 0 .. .. t,) • *' 00 0 0. " 0 tt a ... :10 • • 'IiIIo 41: • 10 • • Jt It 4l' • 't) 3 • .0) G if • 3 (iI • • • 'D .. 43 LIST OF FIGURES FIGURE PAGE Developmental stages in the life history Sapro~ia. of sp... .. •• 1& . 19 2. Map of Pleasant Prairie Township indioating looation of sampling sites . 21 Sampling s1 te on Mahoney·s Creek ...... 21 Sampling site on Mahoneyts Pond ...... 2) Sampling site on the Des Plaines River ...... 23 Sampling site on Ba.rnes Creek ...... 25 7", Sam.pling s1te on La,ke Mi chigan 2.5 8. Traps used to isolate aquatic oomycetes 27 9. Traps showing placement of the covered jars in the Styrofoam 27 10. Mycelium, sporangia and oogonia of SaErolegnia ferax, X61 36 11. Mycelium, sporangia and oogonia of Sapro~egnia fe~, Xlii 36 12. Zoosporangia of Saprolegnia ferax showing two encysted primary zoospores t x460 .. ~...... 38 13. Mycelium, sporangia and oogonia of ~prolegnia ~1clinat X75 Q.~ ~ 38 14. Zoosporangia releasing zoospores of ~apro~egni~ ~1c11na, X18? 40 1.5.. Oogonia. ~ri th antheridia. of Sa.:proleg~ diclina:, X22Q o$ 4) .. .. • • • .. 40 16. Zoosporangia of Dict~lChus ap. showing zoospores X909 42 LIST OF TABLES r~LI\..BLE PAG:E 1 ~ Distribution of Oom_ycetes 29 2. Cornparative reproductive s trueture s «" .. ~ .... " " " " .... " " " ~ " " ... 31 INTRODUC TION Pleasant Prairie Township is one of eight tc)\vnships located in Kenosha County. It is bounded on the East by La.ke 1\.1ichigan; North by 60th Street; Vie st by Highway 1·- 94; and South by the Illinois V1isconsin state line.. The To\vnship covers an area of approxirnately fifty-six square n'liles of low, flat land prirnarily used for farm.ingu Located within this area are several small spring-fed creeks, the Des Plaines River and one spring-fed pond.. This investigation will include five san1.pling areas.. Three of the five sampling areas are basi.cally surrounded by farIn land, the other t\.-vo by unused land with SOTne residential units nearby. The first saITlple area \vas on the North side of lv1.ahoney 1 s Creek; the second was on the North side of Mahoneyfs Pond which drains into J\.lahoney l s Creek; the third \vas on the vVest side of the Des Plaines River; the £olJ.rth along the South side of P,arnes Creek; and the fifth \.-vas on the East shore of Lak.e 1YHchigan. rrhe purpose of this study is to investigate the types of aquatic Oornycete s com.nl.on to the surface layer of each saITlpling site. in these areas by bi-\iVeeh:.ly intervals during the months of August, SeptcInber, October, Noveluber, April, .L\/lay and J'une~ LITERATURE REVIEW .l\ccording to "Volf and W'olf (l947), if fungi are arrlong the Il s iInplest of organiSl1'.ls" their classification should not be too difficult. The stu.dent, however, is forced to conclude that these organisDl.s are far fron"'l si!Ylple. If siln.plicity was the key characteristic the nurnerous volumes vvritten need not exist. All classifications are lllan-Dl.ade and therefore contain flavvs .. The ideal clas sification has not been, nor shall. be, proposed until researchers reveal ITlorphological sirnilarities and differences behveen species and indicate the'il' evolutionary position and geneological relationships nVol£ and W'olf, 1947)" During an addre s s to the Mycological Society' of Anlerica, Go "\V .. Martin (1955) proposed this question, llAre fungi plants? II After an extensive revie\.v of the literature he could only advance reasons for differences of opinion and prolTIote the idea that though differences exist the roadblocks vvhich harnper the progress to the phylogeny of the fungi be rernovedo The clas sification of organisIns Ln.to the tvvo kingdorns, Plantae and ""I.\n:p..irn.alia using the binolTIial sys ten"). sterns frarn the \V ork of Linneaus to fIaeck.el, vvho proposed in 1866 a third kingd()~m Protista which vvas to include all organisrns not clearly plant or ') .) anirnal.. This has been expanded by Copeland in 1956 to a fourth grouping, the :tv1onera (.l\lexopoulos, 1962) which is advocated by il1.any rnodern authors in their general biology texts (\Veisz, 1967; Curtis, 1968; and the BSCS staff 1960, 1968)0 The fungi, irregardless of the major taxa to \vhich they are placed are classified in \vhat appears to be hvo major schen1.es depending 'Lipan the source used.. .A.lexopoulos (1962) classified the fungi into Division Myeota, with hvo sub-divisions; the MyxoDlycotina and the E111nycotina.. In his scheme he gave the group of organisrns under study, the OOD"lycetes, the rank of class.. I-Ioyvever,W'olf and 'Volf (19471 Bessey (1935), :F'itzpatrick (1930), :Langeron and VanbreusegheYn. (1965) and Sparrow (1960) rank the Oorr~ycetes as a subclass in the class Phycor:nyc etes. The OOlTIyceteae are divided into two to five orders depending upon the source used.. ~4...1l au.thors consulted recog.nize the Saproleg niales and Peronsporales with Fitzpatrick (1930), Bessey (1935L \"1"01£ and Wolf (1947) and Langeron and VanbreuseghelTI (1965) including the lYfonoblepharidales, \vhereas, Alexopoulos (1962) includes this group in the class Chytridiolnycetes. Other orders that have been. va rionsly included in the Oornyceteae are: LeptoD"1.itales (Wolf and W'olf, 1947; SparYOVl, 1960; i\lexopoulcls, 1962; and Langeron an.d Vanbreu.segher:n, 1965; Pythiales, (\Volf a.nd vVolf, 1947); and Lagenidiales, (Sparro~w, 1960; and Alexopoulos, 1962)~ Basically the OOTnyceteae consist of those fungi that reproduce 4 asexually by biflagellate zoospores that are borne in sporangia of ... J various types, except i.n the most advanced species in 'which the sporangiurll assurnes the function of a spore and genninates directly by a germ tube (Sparrow, .1958, 1960; l\lexf.)poulos, 1962; .Langeron and Vanbreuseghem., J 9(5). Sexual reproduction ranges from. the entire gt".:i..Illetangiurn being £orrned frorn the whole thallus to heterogarny. The forHlahon of oospores is characteristic of all but the lTI.Ost prin.1.itive species. The oospores originate and ITlature within the oogonia (Sparro'vv, 1960; Alexopoulos, 1962). Structurally, this class consists of organisrns that range £roYl'1. a prirnitive unicellular thallus to a profut;ely branched, fila rnentous rnyceliuYl'1.. The majority of the Oomycetes are eucarpic 'with the more prin1.itive being holocarpic. These organisHls are generally saprophytic, though there are Inany that are parasitic \vith the highest forms being terrestrial obligate parasites (Sparrow, 1958, 1960). The re are tvv-o generally accepted n1.ethods of identifying the Phycon1.ycetes: the older method used by .Fitzpatrick (1930) consists of examining the n'lyceliurn, the thallus, and then the sporangiuITl and galnetangiuill.; the newer n'lcthod, vvhich is a distinct departure, estab··· by Sparro\v (l958, 1960) involves exaroin:ing the flagellation of the zoospores, then the thallus and finally the garnetangiurn~ 5 'There are several exalTIples of a l1typicalH life cycle for the Phyconiycetes in general and the OOInycetes in particular. R.aperfs (1939--1951) classical investigation of the sexual ll1.ech aniem of Achlya bisexualis Coker and Achlya ambisexualis Raper is heavily referred to, ho\vever, the dioecious condition is not prevalent enough in this class to consider it a representative of this class (Figure 1). In the soulatic portion of Saprolegnia, the thallus is cOluposed of t\vo types of hyphae; the rhizoidial type found in the substratum.; and the branced hypha.e that forrn the visible colony, \-vhere the repro ductive o:r:gans a.re found. Depending upon env'ironrnental conditions, either the sporangium or the galuetangiull1. \vi11 appear nVolf and -WolL 1947; Elliott, 1968; .]Vioore, 1968). The zoospores derrH)nstrate diplanetism while the oogonia, \vhich contain one to n1any oospheres, will form thick \valled diploid oospores (Couch, 1941; Sparro'vv, 1958, .1 960; Alexopoulos, 1962)• .Ecologically, few specific studies have been done. Infor ll1.ation is uSllally gained from. the accounts of the n1.orphology, taxonoruy and physiology of specific fungi. The area of pollution and environ.1uental studies has given ne\v in1.petus to the study of fungi in water systems a,nd should ad.d ecological inforr:nation concerning the Ph.ycOITlycetes. The qualitative con1positioD of fungal populations, the substra.ta, a.nd their place and role in the aquatic en\lironHH:.'.nt is 6 fairly well known. vVhich organisrns and their precise functions in degrad ation of. o:rganic rnaterials is less \\1811 kno'vvn (Sparrovl, 19(8)0 Quantitati'vely the re is ve ry little knowTl about the PhycaH1Y ceteso l-Iov\f significant and effective they are in decornpostion; how n1.any organislll.s exist per given volur-ne; their distribution, both horizontally a.nd vertically as to stratification in \vater; their relationship and nurY:l ber in the benthos is not knov;,rn (Sparro\Jv, 1968)~ The aquatic Phycom.ycetes can be found in pools, lakes, ponds, rivers, strearns, estuaries and other marginal areas in some form .. To date no fungi have been isolated fron1. thernl.al springs, though sphagnunJ bogs 'vvith their low pH support a rich and diverse variety of Phycomycetes such as :::0--chlya, Pythiurn and Phlyctochvtrium (Miner, 1965). Thraustoc1:.ytriuln has been found in salt lakes (SparrO'w, 1968) a.nd Saprolegnia and Achlya in acidotrophic lakes (Suzuki, 1960). Factors found in the habitat that a.££ect these organislXls to SOUle degree are: turbidity; light; substrat:uD1.; tern.perature; ox.ygen, pI-I; altitude and seasonal change s 0 The a.:rnount of solid luaterial in suspension can affect the distribution of the organisrns and substrate availability., Light affects these organisnl.s indirectly through tem.perahlre, algal growth on substrata, and in some cases, the reproductive cycle, i. e., Bla£-3to cladie~~~ em.ersonii (Cantino and I-Iyatt) a.nd 1?lastocladiell~~.1~ritanica ~H()renstein and CantinQ; (I-Iorenstein and Cantina, 1961) .. 7 Subs tratmTI availability and ch.aracter are irnportant to heterotrophs~ It has been found that rnany Phyccnnycetes are selective in the laboratory (\Nlriffen, 1941). It has been found that the zoospores have a chernotactic ability and that they can be affected by age and solutes available (Sparro\-v, 1968)~ Tenl.perature directly affects gern1.ination, vegetative gTov1lth and reproduction. Tenl.perature ranges vary behveen organisms, but in general, fungal structures have been found functioning bet\veen. six degrees Centigrade in Blastocladia (Cotner, 1930) to twenty-five de grees Centigrade in i\chlya_ (Reischer, 1949).. The relationship of teITlperai-ure to the induction of rnorphological structures of taxonoD1.1c irnportance has been shown to be a pronrising new area of investigation (Sparrow, 1968). Aquatic Phyconl.ycetes vary greatly in the a:mount of oxygen needed for nutrition and growth.. It has been shoviln that cel:tain specie s of Apodachlya and Sapromyces , require an oxygen- rich en~ ·vi.ronrnent and s orne specie s of Blastocladia a~(ld Rhipidiun1. \vi11 groVv where there is little oxygen present and the environm.ent is foul, de pending upon the particular rnetabolisl1:1 of the organisl'll (Enlerson and Cantina, 1948; Sparravv, 1968)" ~Aquatic fun.gi have been found in ha.bitats ranging in pH fronl 1 .. 9 tc> 8$ 0, throug~h the gene ral range is a. pI-I of 5 .. 6 - 7 .. 4 .. l\quatic pI-I cannot be considel~ed by itself but lTIUSt also be considered in re lation to the dis solved carbon dioxide and bicarbonate v./hich play an 8 ilnportant role in p:H change (Roberts, 1963).. It has been derYlonstrated that the aquatic fungi follc}\v a season: al occurrence with spring~ as the most favorable season for bo-rowth.. During the SUmlll.er, when the ternperature increases, \vater levels fall and pH decreases, the Phycomycetes show the greatest decline .. In the autumn, \vith the advent of lo\ver telnperature, more \vater and an abundance of substratuITl, there is a renevval of gro\vth (Sparrow', 1968) .. There is little evidence of ecological succes sion though it has been conunonly assU1TI.cd to occur. Also, it is not knov;,rn Vilhether species that occur at the saIne tiIne forJu cOlTI1TI.unities or are just aggregations of indivriduals (Sparrow, 1968). Much work and investigation needs to be done in this area in order to fully understand the life patterns of the aquatic Phycolnycetes. The c.lass OODl.ycetes which includes the water luoId, \vhite rusts and do\vny rnilde\vs have rather diverse habitats (Alexopoulos, 1962)~ The Lagenidiales com.prise a group of prilnarily lll.icroscopic, endobiotic parasitic fungi that are found in fresh and rnarine water .. Specie s of Q).pidio}?sis and Rozellopsis for exaln.ple) are parasitic on. aquatic Phycomycetes and several algal forms. Very little \vork has been done to ascertain the cOlnplete range of hosts 'v\lhich these fungi Vvill attack,(Sparrow, 1960). 'T.'he Lepton:u.tales are the only group that have been foun.d solely in fresh "'\,:\rater as saprophytes occurring c>n vegetable debris.. They are o / found in both cool, clear \.vater and lJ.na.er exceedingly polluted con ditions (Sparrow, 1960; vVilloughby and Collins, 1966) .. They differ {rom. the other Oornycetes in that they are regularl)'" septate, and except in .i\podachlyella. cornpleta, contain only one oosphere (Sparrow', 1960).. The Peronosporales comprise a primarily terrestrial, microscopic fungi parasitic for the rnost part on flowering plants .. The Pythiaceae is the only fanl.ily that displays a range of habitats from. aquatic to amphibious and terrestrial.. It is felt that the nl.ajority of these organisn"ls are able to lead a saprophytic existence.. They are generally identified by the characteristics of the sporangia and the sporangiophores.. PythiuITl sp .. is an exarnple of the sporangial type and !:~yonospora sp.. \v0uld be an exarnple of the advanced sporangio phorial type (Sparrow~ 1960; Alexopoulos, 1962). The term \7l;rater ITlold is custornarily used to designate the Sapirolegniales.. The ITlajority of this group are aquatic saprophytes, though lnany species are soil-inhabiting and s 01Y.lC are parasitic on both plants and anirnals. Structurally, they range {rorn one-celled) ho1ocarpic organislns parasitic on algae to those with 'well-developed coenocyt:ic, non- septate n"lyce1iilm, , except for diffe rentiatioD. of the reproductive structures. These organis:rns have been found on a. \.vorlcl-v,lide basis .. Because of thei r distribution, ea.s e of growth and. adaptability arnong other items, the Phyconj-ycetes have pla.yed a role in studies 10 of genetic, cytology, taxonomy and rYloTphology.. The oonlycetes have been studied to som.e extent in these various areaSljl Cu.rrent literature covers topics such as genetics, where it has 'been fuund that ITleiosis does not occur in the gerrninating oospore (Ziegler~ 1953) but rather in the ga.metangia (lv1u.llins, 1968).. lv1:ullins (l 968) als 0 found that wate r rnolds other than Achlya sp .. had rep roduction initiated. and con trolled h~l horrnones .. Clausz (1968), beside[~ describing a different method of classification based on oospore strllcture, felt that ten.1p e rature and light als 0 played. an ilnportant part on oogonial structure and reproduction. 1:vlost of the work done has stenuned froln the w'ork done by Raper (1940, 1947, 1951) and Barksdale (1960,1962,1963, 1966)" Gleason (1968) has done excellent \vork on the nutrit:ional aspects of the Leptornitales. He has found that in this group there was no single carbon source that was adequate for gro\vth of the organiSITIS tested, however, asparagine was found to tak.e care of the nitrogen requirelYlents of all tested species .. Taxonornically, n.luch "''lark has been done on this class .. Couch (1938, 1941) describes techniques of flagellar staining and descriptions of zoospores that had a bearing on Sparrowl s (1958, 1960) \vork.. Hendrix (1968) has described a nevo,! species of Pythium. based on heterothallislTI and Karling (1968) has added the genus A.phanom.ycop sis in the Saprolegniaceae near Aphanornyce~_as two examples of \york. recently done taxonurnically· .. As a group the aquatic OOInycete s have not been shown to be 11 ecologically or economically irnportant.. Several exceptions to fhis vvould be the fish and crayfish Iuolds (Unestarn, 1968; Stuart and F'u11er, 1968) and sonl.e 0.£ the arnphibiol1S forms of the I)ythiales (Alexopoulos, 1962).. It is felt that the di.stribution of the Saprolegniales is of wider occurrence than just fresh\vater. Dick (1968); Stuart and Fuller (1968) 'Using both literature revie\v and experiruentation beHeve that this group is quite prevalent in mixed saline w"aters of estuaries .. Studies done on the effect of chemical and n"1.echanical agents in an att:ernpt to understand the organisl'n to control it are qllite en lightening.. .Hick1nan and Ho (1966) found that zoospore attraction of the Pythiales can be altered by application of chenucals, urea, grc)\:vth substances and antibiotics. Unesta:rn (1968) found that the enzyrne chitinase of A.phanon"1.yces astaci Schikora \vas affected by the rnetalic ions of absorbtion of chitinase by chitin protected against shaking, heating and pH changes .. 1'viartin (1968) in his esperiluental work sho~ted that :malachite green was effective against various Saprolegniales in dosages ranging froln 5 ppIn/5 rn-inutes to 15 ppn1./hour depending upon the species, \vhereas, acriflavine only started. to becoITle effective at 100 ppnl./hou.r .. Gysan (1969) reports that a chelated organic copper cOlnpound (cutrine) de'\Tcloped by Dr .. B .. DOHlogalla efiectively controls Sapro1.egn~~ Sp .. with no fish loss when applied at t pprn in a drip systen.1" Barrick (l9(9) reported that copper sulfate used as a fungicide v;;-as deadly to trout at a concentration as 10\V as 0" 14. pprn vvhereas copper: 12 in the fOrD], of an organic conl.pound 'killed fish when applied in excess of t ppIXl coppel' ion yet was effective in controlling Saprolegnia at ~ ppm9 In a separate series of experirnents (RG D. Blackburn unpub lished data) during 1970, it \vas found that Cutrine applied at an equiv ..· alent rate of copper sulfate \I\,ras les s toxic to fish, yet it controlled Saprolegnia. 1\ Collins and Po Brugenl.an (personal cornlllunication 1970) state that the chelated organic copper GOlnpound (Cut1'ine) is effective in controlling Saprolegnia with no fish kill vvhen applied at the reconuuended rate of 2 .. 2 pprn/ac1'e .. W'illoughby (.1·968) comnl.ented that non- oogonial farIns of Saprolegnia usually isolated froITl fish constitute a definite prop(n~tion of the rnicroflora of aquatic areas. 'The significance of this to the en vironrnental make up of fresh water should not be undc:resti!uated" Further adequate methods of control should be developed to prevent the los s of fish populations to help nl.aintain a proper ecological balance. 'With m,ore research 1n the physiology of the aquatic Phycomycetes, their relationship to the total ecology should_ becolne apparent.. lYii\ TERIA.LS i\.J\JD lvfETIIODS DESCRIPTION OF TIlE S.l\.1v1PLING AREAS The five saulpling areas are located \vithin Pleasant Prairie Township \·vhich. is located in the Southeast corner of Kenosha County, \Visconsin" Each sampling area is identified on the" map of Pleasant Prairie (Fig .. 2). M.ahoney1s Creek is a very narrow, shallo'\v, Ineandering creel., that flo'ws Eastward from 1'v1ahoneyts Pc>nd to 34th i\venue '\vhere it empties into a storm sewer" Its width varies fron"1. two to four feet and the depth varies fronl six inches to two feet.. The bottonl is COIT1 posed prirnarily of sand, gravel and silt. 'The vegetation on the creek bank is composed of cattails, Saggetaria, sedges and willo\.vs.. 'The \.vater contains an unidentified filamentous mat of algae attached to debris .. The creek flow's through an area 'where the banks rise to eight feet in height at the point of entrance into the stoYxn sewer.. The sanl:ple area is approxirnately one hundred yards west of the storm se\-ver. In this a.rea the creek is fifteen inches deep with eight inches of silt on the bottom.. It is seven feet below the norrnal ground level at this point.. The \vater is usually silty and nloves rather slowly (Fig. 3) .. JVlahoney 1 s Pond is located \"vest of 39thAvenue, south. of County Roa.d EZ, east of 52nd Avenue, a.nd north of 89th Street. It is a spring- fed pond about 500 yards long and 60 yard.s \-vide w'ith an average depth. 14 six feet.. The pond has a narro\-v, shallow' shore line that drops off rapidly three feet frOln the shore at the sampling site.. The vegetation in this area is com.posed basically of \vil1ows and cattails along tb.e shore .. The \;l;,rater appe.ars to contain a la.rge a.m.ount of silt in sus· pension at all tilnes. The san'lphng site is loca.ted 150 yards \-vest of 39th .t'\venue on the north side of the pond.. The water exhibits no noticeable movement except ~Nind ripples (Fig .. 4) .. The Des Plaines River is a long, slc)\v-moving shallo"w river that begins in Somers TOVvll ship, Kenosha County and flo\vs s ol.1theas te rly through Kenosha County to eventually empty into the Illinois River near J oliet~ I.llinois. At the salllpling site, the rive r is norlllally sixty feet wide and three feet deep at its deepest point. The sam_pIing point was just south of lligh\,vay G" This area changes in depth and \-vidth "when ever there is a lTI.easurable arnaunt of rainfall (which con'lplicated the san1.pling procedures).. The bottorn of the :rivex is silty" The main vegetation in the area consists of w'illo\vs and cattails (Fig .. 5) .. Barnes Creek is a narrOV'l, rather straight creek approxirnately one-fourth. mile long that begins in or near a marsh east of Highw-ay 32 and empties into I-iake ~/1ichigan.. It is located half-\.vay betv/een 116th Street on the south and. 104th Street on the north~ The creek fOrlYtS a pond before entering Lake Michigan.. The sampling site is loc.ated twenty yards east of Fil'st Avenue along the south side of the pond~ The pond area is 30 yard;.:' 'Nide a.nd fi've feet deep at this point" The bank. of the 15 pond is five feet high and covered with poison ivy" The edges of the pond are norn'lally covered ';,vith Cladophora; the \"later is clear, and botton1. is sandy. The water Dloves slowly toward the lake (Fig« 6). Lake lv1i.chigan forms the entire eastern boundary of Pleasant Prairie Tov;rnship. The san1.:pling site \vas located t\venty yards north of the rnouth of Barnes Creek~ At this point the lake has a rocky, sandy bottom \vith a]..TI1ost oonstant \vave action u.pon the beach.. The water contains sedin1.ent, usually sand.. A sandy beach is directly adjacent to the lake (.Fig .. 7) .. PROCEDURE W'ater saluples Vi/ere collected in traps froln each area on one to two week intervals fron1. August 13, 1969, to Novenlber 18, 1969, and .April 25, 1970, to June 12, 1970. A total of 12 \vater san.1.ples Vi/ere obtained.. Traps were usually set out and picked up on \tveekends .. Norn1.ally, if there Vi.TaS no growth apparent in the traps, they \vere left until the next pi.ckup tin-le" Sanlples from. each area we re collected to determine \vhich aquatic Oon1.ycetes were present.. Ten sInalJ. baby £0("H:1 jars \vere used as collecting cham.bers .. ~;\fter the jars \vere sterilized, five were baited vvith boiled hernp seeds and five \vere baited \-vith dead flies.. Each jar 'was covered \-"lith a three inch by three inch piece of #12 bleached cheese cloth 'which vv'as secured \vith a five foot length of heavy corel. This was a modification of the rn_cthods used by Patterson (1967) and A .. C .. Nelson (personal COIT.tIl1uni cation 1969). 1 / .l. tJ Before placing the traps in the study site they V,fere filled one-half full of water fror.n the siteo The bottles vvel'e then placed in the\va.ter and inverted so that the open end \vas in contact w'ith the \vate r of the sarnpling site" The jars 'we re left at the sa.mpling site for periods ranging froln one to hvo ·weeks. Im.D"lcdia,tely afte T collect ing the traps they were placed in the investigators baserrlent (18°C) for continued incubation. At the tinl.e of the first collection period many of t11.e traps \vere found D"lissing or nlouth up. In order to rectify the situationt the in vestigator devIsed floats m.ade of 3/8th inch styrofoan1. cut into four by six inch pieces,. TVlo holes, the diallleter of the jars, 'were cut in each \-vith a hole punched in the end for the cord (Fig" 8 & 9)" The floats \iVere placed at each. saITlpling site 'with one trap containing henl.p seeds and the second containing dead flies. Each trap was fined one~half full of ~late r frOIn the site and inverted so the mouths vvould be in con tact \vith the ',V"ater.. Exeellent results vvere obtained, except in cases \vhere the v'later level rose or fell drastically. Each seed and fly \vas exanl.ined under the microscope at lOOX to deterlnine if there \Vas. any fungal grovvth present.. If so, each 'Nas exarnined to dete rrnine vvhat ty-pe of zppsporangial, oogonial and hyphal structures \.vere present in order to identify the organiS111 according to Sparr()"w (1960) and Gilman (1957). The investigator tried several stains and fixatives to aid in identification but they caused distorticHl of the orgarrisrD and atternpts 17 to e:rnploy then1. were unsuccessful.. Collections ,vere not made during the n1.onths \vhen each body of wate r had an ice cover; hovvever, one gallon sarnples \vere collected and baited and left at roonl. ten:lperature, along ,vith thos e from. the traps collected in late November. 'W'hen gro'wth appeared the baits 'were ex arnined as previously stated to deternl.1.ne the organisll1 present. In order to obtain pure cultures of the Oornycetes grov:,ring on the baits a standard corn meal agar media Vlas prepared by using 17 g .. of Difco Corn lv1eal Agar and 1000 ml of distilled vvater.. 'l'his rnedia \vas sterilized for 15 rninutes a.t 15 Ibs pressure.. '1'he cooled rnedia ,vas poured into pre-sterilized plastic petri dishes and allovved to solidfy. Sterile glass slides, three inches by one-half inch, \.vere placed. upright into the solidified agar through the cente r of the plate, d.ividing it into tvvo equal halves" Each plate was inoculated by centering a sn1.all portion of the bait frOITI each trap one-fourtlh inch in frorn the slide on one side only.. The innoculated plate s \ve re allo\.ved to incubate at 21°C to sirD.ulate normal water telnperatures 0 'Alben good lTI.ycelial gro\vth appeared on the opposite side of the slide frOHl the point of innoculation the nlycelial material "\vas aseptically transfered to corn ill.eal agar slantso The slants ,vere allowed to incubate at 21°C until good gro\vth \'1:,/<-18 apparent at \vhich tim.e each culture was exalllinedll If one is interested in obtaini.ng and maintaining pure cultures, lv1iller (1967), Wolf (1942), Dick (.1965) and Barksdale (1962) have rnethods vvbich appear to be satisfactory for this purpose" Figure 1. Developluental stages in the life history of SaErolegnia sp. 19 Gerrnination ':'';;;'i·'~'fsecondary ~,t zoospore F "'fhv G~ Encysted ~ zoospore .,....0\ 'i!:" H ii~ " ' •.:~ JJ"t:{. Germination ': ',; Penetration of Fe rtilization Tube Figure. 1 Figure 2. 1vlap of Pleasant Prairie To\vnship iudi eating location of sampling sites Figure 3. Sampling site on Mahoney's Creels: Fiourec 2 (: I 1 :~ r .Figure 3 Figure 4. Sampling site on Mahoneyl s Pond Figure 5. Sarnpling site on the Des Plaines River jl) l HI Figure 4 J UL I G .F 1grlre 5 .Figure 6. Sarupling site on Barnes Creek Figure 7. Sampling site on Lake J:v1ichigan 25 Figure 6 ,J Ul ; n Figure 7 l"igure Sa Traps used to isolate aquatic Oo:rnycetes Figure 9. Traps showing placen:J.ent ci covered ja.rs in the styrofoarn 27 J Ul ) 0 .F'igl1re 8 ! li ~ ! I) Figllrc' 9 28 RESULTS A total of five species of OOITlycetes '~Nere isolated a.nd iden tified; a 1v1uco.rales and a Chytridiales v,,'cre also found" Of the five 00lTIycetes, three \vere rnenl.bers of the Saprolegniales, one Leptcnn itales and one Personosporales.. The Saprolegniales identified were: Sapr.21.~gnia {erax, Saprolegnia diclina, and Diety-uehus spo The .Leptolllitaies \vas Apodac~ella cornpleta and the Peronosporales was a PythiUD1. sp .. The ITlost frequently isolated and identified species \vas S .. ferax. It was found in all sarnpling sites, using both flies and henl.p seeds, except Barnes Creek.. Pythillr.n sp" w'as identified at t\vo locations .. It "vas found using the flies at Barnes Creek and the hemp seeds at Barnes and Ivla.honey's Creek (Table 1 ) .. S .. d:i.clina wa.s identified, using flies, at three locations- Barnes Creek, Lake l.vlichigan and Des Plaines River.. Dictyuchus '\vas identified, using flies, at 1'v1ahoney1s pond and Lake Michigan.. l\.o COITl: £.leta was identified only at one site.. This site "vas Lake Michigan and heulp seeds Vlere used as bait (Table 1)" The types of Oomycetes clid not vary significantly '\"lith the 1 ocation.. S .. ferax \Vas found in the still waters of Mahoney's Pond, the running vvaters of 1v1ahoneyts Creek and in the turbu.lent \Vave action waters of Lake Michigan.. It appeared to be universally distributed .. Only hvo species \-vere isolated frorn still pond water-- Dictyuchus and S" ferax.. Ilo\vever, both VJere also found in I.lake IvHchigan9 ...... _-,,, _-- SITE ORCr"A.NISrv1 .A"pod~chyella Saprolegnia Sa ;prolegnia Dictyuchus Pythiun1 c:..?n1pleta ferax diclina lV1ahoney Creek Fly + Hern.p + 1vlahoney P and Fly + I-Ielup + Des Plaines River Fly + IIernp + 'Barnes Creek .Fly + + I-Iernp _..._------_.__.._._---~._------Lake :Michigan Fly + + Hen1.p + + _._._--_.~-_._-_.._._.._~ ..._------ ----_...•...._.. ..._----- __ ['-.J Table I. Distribu.tion of Oon1ycetes '-0 30 Pytbium "vas isolated from. the creeks only an.d not isolated £raIn the rivers, ponds or lake.. This species \vas identified fron1 Barnes and Mahoney!s Creek~ So diclina and the Mucorales were isolated from both creek vvater and the lake.. j},;.o cOlnpleta vvas isolated froDl. lake water only .. There did not appear to be any effect on the nun1.ber of colonies due to seasonal changes nor did there appear to be a Inajor difference. in species by season in this studyo The number of species found in each v;.rater sarnple did not differ significantly with the use of either heDl.p seed or fly as a sub stratum.. There Vi/ere nine Oonlycetes isolated using a fly as a sub strate and seven OOlnycetes isolated using hen1.p seeds as a substrate.. Each species was identified 'by Dl.eaSu.rernent of their vegetative and reproductive organs and associated characteristics as presented in Gilm.an (1957) and Sparrow (1960).. Table Z contains both the expected and observed characteristics and :measurements as a source for COITl parison.. ORGi\.N ORGANIS.M. Sa·.Erolegni~ Sapro1egnia Dictyuchus Apodachyella P ythiU.1T!. ferax diclina COPJ.p1.e ta.. ..9.i~_g 0 nia ~Expected 37 - 97>~ 35 - 1 00 -- 1 23 - 48 --- 1 Observed 81. ~'4>;( 66 I 36 J. .Q_~_spore E~xpected 24 - 30 20 - 26 -1 18 - 24 -1 Observed 25 22 - I 18 .1 ZoosE?rangi~ Expected 2 -- 2 14-36x500 .3 ZO - 30 Observed 37 x 300 .30 x 400 25 x 250 --- 3 30 Zoospore Expected 9 11 - 1.1+5 11. - 16 --- .3 8 - .1 0 Observed 9 II 11 - 13 .3 9 >;: all units are in Dl-icrons 1 SCHIle species do not ShO\iV oogonia (Gil:rnan 1957, Sparro\v 1960) 2 Sizes not given in Sparrow (1960) or Gilman (1957) 3 A.podf::_~hyel1~ ~pleta does not: hav'e zoosporangia.. (Sparrow 1960) -----_.._------_._------_.._~---_._--_.- --_..----- !..,;.) Table II. Conlpa:rative Reproductive Structures I-< 32 DISCUSSION" l\.s the purpose of this study "was to isolate and identify the OOITlycetes found in these five saITlpling sites in Kenosha County, it J is necessary to describe the characteristics by which the species ,Xlere identified .. Saprolegnia ferax had hyphae that were m.oderately stout and sparingly branched (Fig~ 10, II). There \-vere many sporangia that \vere ,-vavy and often tapered upward; the zppspores ,;vere generally 9 Ul1crons in diam.eter (Fig.. 12).. Very few genunae \vere found w'itbjn this species.. The oogonia were l1Ulnerous and varied in size (see Table 1) ,-vith '"valls 1-1 .. 6 microns thick "\Nhich contained pits.. The oogonia ,-ve re not usually found in chains but ,-ve re te rlninal and sOlnetimes intercalary.. Oospores \vere centric and nUlYlbered froln 1-20" Antheridia \-vere rarely found in this species ( Fig .. 10, 11 ). Saprolegnia. hyphae (Fig. 13). SOIne of the Sporangia \vere enlarged and broad near the end" Spores \X,Tere 11 m.icrons in diameter (Fig .. 14). S~ diclina is diclinous \vith oogonia and anthe.ridia being produced on the sam.e rnyceliurn (Fig. 15). Oogonia \vere found in chains and 'Vv'ere inter calary as v,,~el1 as terrnina.L A.ntheridial branches arose diclinously fro:r:n near or distant hyphae and sOlnetirnes covered the oogonia. Oospores ranged in size (Table 1) and nurnbered £r01TI 2 - 20~ 33 Vegetatively Dictyuehus is similar in appearance to.L<\chlya. T'he sporangia \vere cylindrical, blunt and borne in a zigzag pattern.. The sporangia showed a strong tendency to fall a\vay and lie free in the water prior to spore escape" The spores rernained in the sporangia forlning a network and eventua.lly elnerge by individual openings to the outside as secondary zppspores" The oogonia \vere spherical and sUlooth \vhen present. Oospores \vere one to Dl.any but didn It fill the oogonia (Fig" 16).. Apod?-chyella con"1.pleta \Xlas conlpos ed of s lende T, filarnentolls 3 irregularly branched rnycelia containing pseudosepta.. Zoosporangia vvere not observed in this genera. A. cOlnpleta differs frorD. Apodac~ by having rnc>re than one oospore per oogonia.. The oogonium. \Nere spherical, tern1.inal and single.. The oospores contained a large ec centric oil drop and \vere approxinlately 1 b rn.ic rons in dianlete r. In Pythiuln sp. the 11:1yceliunl vvas 'wel1 dev'eloped consisting of rnuch branched hyphae \vhich forr-n.ed tangled com.plexes~ The zoospor angia \vere filanlentous and sin"1.ple.. Zoospores were biflagellated and. reniform" Oogonia \Vel"e not. ob served ~ There \vere lirnitations to this study of the Oornycetes.. The prilne lin"litation is that this is a taxonomic study only, and no atternpt \vas n'lade to correlate the quantity of Oornycetes with season, telnperature, pI-I or location" The results therefore are valid as organisms \vere isolated and identified for each location• .:.'4.. limitation at each location 'Nas that only one site \va.s chosen and this particular site \~laS used throughout the study. A n'lore COIYl 34 prehensive study on distribution should have included more sites at each area at spaced intervals to deterlnine Oomycetes over a broader area and changes \vith location and current in each area~ Ho\vever, in a study done by Paterson (1967) all. the aquatic habitats of Phycomycetes in Northern lviichigan, it vvas found that there vvas no distribution pattern. It Vias noted that he found. SQ ferax and S~, di~lina, alllong other organislns in Lake Iviichigan and Douglas Lake using h.em.p s ceds as his Hlost effective bait.. A limiting problerD of tIlls study \vas the characte r of the water itself.. The investigator was han'1.pered rnany tin'1.es £rorn collecting sarnples by the great variation in \vater level due to rainfall or the lack of it and the turbulence of the \:vater due to 'Yvind speed and directionQ _A.. technical problem faced was the use of the stain~ Lactophenol cotton blue.. The stain, which \vas intended for use as a stain and preservative for the organis1TI, instead distorted the shapes of the re productive organs and m.ade the organiSD'1 even IT10re difficult to identify.. Figure 1. O. 1v1yceliurn, sporangia and oogonia of SapJ;olegnia ferax, X 61 ~ Figure 11 ~ Myceliulll, sporangia and oogonia of Saprolegnia fea--ax, X 11.1 • 36 Figure 10 J:.:: if) Figure 11 Figure 12. ZoospoTangia sho\ving t\v'O encysted prin1.ary zoospores, X 46D . .~F'igure 1 3. M ycelil.11Yl, sporangia and oogonia of Saprolegnia dj_~lina , X 75. ~) Q .)0 Figure 12 Figure 14_ Zoosporangia releasing zoospores of Sap:r()leg~ia diclina, X 187. Figure 1:5. Oogonia vvith antheridia of Sa12rolegni_~ didlna, X 220. 40 ,I ~: i'D "Figure 14 Figure 16. Zoosporangia of Dictyuchus sp. showing zoospores, X 909. .Figure 16 SUlv1MARY The purpose of this study Vilas to deterrnine the types of Oon1.ycetes present in aquatic habitats in Pleasant Prairie TOV1D Sillp. These organisIns ·were collected \-vith the use of traps and then cultured c>n agar to obtain pure cultures. With the us e of high Inicxoscopic Hlagnification reproductive structures could be observed and identification could be Inade. Pictures and photomic· rographs were used in recording san1.pling sites and illustrating various reproductive structures. 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