Applications of the vital fluorescent labeling technique with brighteners to studies of saprophytic behavior of Phytophthora in soil

Soil Biol. Biochcm. VoL 2, pp. 247-256. Pmgamon Press 1970. Prinbadin Great Britain

APPLICATIONS TECHNIQUE SAPROPHYTIC

OF THE VITAL FLUORESCENT LABELING WITH BRIGHTENERS TO STUDIES OF BEHAVIOR OF PHYTOPHTHORA IN SOIL P. H. TSAO

Department of Plant Pathology, University of California, Riverside, California (Accepted 18 March 1970) Summary-The fluorescent brighten&, ‘Calcofluor White M2R New’ [&odium salt of 4,4’bis(eanil~yla~~~-~iazin-2-ylamino)-~~~til~~~fonic acid] at 100-300 ppm was effective as a vital stain in lab&kg mycelia and various spom forms of Phyzo~hf~u spp. Labeling was achieved by growing the fungus in a liquid medium containing the brightener or by direct staining of fungal propagules before use. The brightener was readily absorbed by the fongus and was non-toxic at the concentrations used. Saprophytic behavior of P. purasitica in soil WBS stdied by introducing labeled propagul~ directly into soil and recovering them periodically for observation using a soil-smear technique. Under the fluorescenoe microscope the labeled propagules and subsequently formed germ tubes, my&a, chlamydospores, sporangia, and encysted zoospores all fluoresced with little interfexenoefrom tbe soil mass or soil mkro&a. Spore germioation, myalial growth, sporuiation, and lysis of P. pmz&ica in nonwded and amended natural &Is have been studied with this 1abeIingtechnique. Labeling was successful also with several other soil fungi. iNTRODWXION

THEslow progress in soil ecological studies of ~~yro~~~~~~spp. and many other soiLborne plant pathogenic fungi can be attributed, in part, to the lack of adequate methodology for such studies. Due to the opaque nature of soii, difficulties arc encountered in studying the saprophytic behavior of these organisms in the soil environment. Consequently, workers have resorted to indirect methods which include the use of soil extracts, soil diffusates, or incubation teclmiques involving substrates which contain nutrients. Although these methods generally provide valuable information, their shortcomings were adequately discussed by Lockwood (1964). Few reports on studies of fungal spore germination and mycelial growth in soil employed direct methods which allowed an intimate contact of test organisms with the soil without the involvement of exogenous nutrient factors. In these methods, the fungal propagules were ad&d directly to the soil and, after incubation, were recovered for observation by plastic 4%~ (Lingappa & Lockwood, 1963), by agar block or agar strip for making a spore print (Boosalis, 1960; Jooste, 1963, by preparing a soil smear slide (Nash ef al., 1961) or by placing recovered soil suspensions on agar (Papavim 1967). The recovered propagules are usually stained with common dyes for ease of recognition. The methods generally work well and provide accurate information on the true nature of the behavior of the test fungi in soil, but also have limitations. The tit fungi are often morphologically similar to some other fungi naturally present in the soil. The problem is inter&f&A when the soil is amended with organic nutrients which stimulate a profuse growth of all microorganisms. Soil particles on recovery substrates can also interfere with the detection and observation of the test organisms. Darken (1961) reported the use of fluorescent, ultraviolet-absorbing brighteners in microbiological studies of several actinomycetes, bacteria and fungi. The vital stains were 247

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non-toxic, and were readily absorbed by the microorganisms studied. The labeled propagules and the subsequently formed structures were fluorescent when viewed with fluorescence microscopy. The method has since been applied to other biological systems (Cole, 1964; Darken, 1962; Darken and Swift, 1964; Harrington and Raper, 1968; Paton and Ayers, 1964; Patton and Nicholls, 1966; Tsao, 1969b; Wilson, 1966). This paper reports the applications of this versatile technique to soil ecological studies, with major emphasis on Phytophthoru spp. A brief account of the study has been reported previously (Tsao, 1969a). Since the initiation of this work in early 1966, a similar study has appeared (Eren and Pramer, 1968). MATERIALS AND METHODS

Fungus species and isolates Phytophthoraparasifica Dast., isolate T131, was used in most of the experiments. Other species and isolates of Phytophthora tested were P. cuctorum (Leb. & Cohn) Schroet., isolates P235 and P283; P. capsici Leonian, isolate P504; P. cinnamomi Rands, isolate SB216-1; P. citrophthoru (R. E. Sm. & E. H. Sm.) Leonian, isolate 1309-A ; P. colocasiae Rat., isolate P355; P. drechsleri Tucker, isolate P209; P. erythroseptica Pethyb., isolate P340; P. heveae Thompson, isolate P379; P. megasperma Drechs., isolate P339; P. megasperma Drechs. var. so@ A. A. Hildeb., isolate P405-1; P. pulmivora (Butl.) Butl., isolates P253 and P441; and another P. parasitica, isolate T89. Other fungi included in some of the experiments were Fusarium solani (Mart.) Appel & Wr. emend. Snyd. t Hans., isolate T77; Thielaviopsis basicola (Berk. & Br.) Ferr., isolate T35; and Verticillium albo-atrum Reinke & Berth., isolate V3. Brighteners

The diamino stilbene brightener, ‘Calcofluor White M2R New’ (M2R) obtained from American Cyanamid Co., was used in most of the experiments. It is a disodium salt of 4,4’ -bis(4-anilino-6-diethylamino-s-triazin-2-yla~no~2,2’-stilbene~sulfonic acid. The pow&red preparation contained 70 per cent active ingredient and 30 per cent diluent consisting of a mixture of anhydrous sodium sulphate and sodium carbonate to improve solubility. Other formulations of Calcofluor White tested were ST, PMS and RW. Sterilization of aqueous solutions of the brighteners was, in most experiments, by Millipore membrane filtration (0~22pm or 0*45pm pore size) of a stock solution prepared at 25 x or 100 x the desired final concentration. Sterilization by autoclaving was not satisfactory because of the presence, in some preparations, of fluorescent particles which remained in the stock solution and interfered with the observation of fungal propagules. Concentrations of the brighteners reported in the paper are based on active ingredients. Production of fungal propagules Axe& cultures were used in all experiments. Phytophthora spp. were grown in cleared V8-CaCOI broth (filtered Campbell V8 juice, 100 ml; filtered 2% CaCO,, 100 ml; deionized water, 800 ml) in 12-0~. prescription bottles. When mycelia were used, the cultures were harvested after 3-4 days of growth in stationary culture at 25°C. For the production of sporangia of P. parasitica, the method of Menyonga and Tsao (1966) was used. Sporangia of P. cinnamomi were obtained by the method of Chen and Zentmyer (1969). Sporangia of other Phytophthora spp. were similarly produced. For the production of zoospores of P. parasitica, a sporangium-bearing mat was washed

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and then chilled for 15 min in deionized water at 18-20°C (Menyonga and Tsao, 1966). Zoospores of other Phytophthoru spp. were obtained after chilling washed mats for various periods at varying low temperatures. When synchronous cultures were needed for inoculation, the zoospore suspension was shaken on a Vortex mixer or reciprocal shaker (Menyonga and Tsao, 1966) to induce encystment. The encysted zoospores were washed by centrifugation three times before use. For the production of chlamydospores of P. parasitica, a modification of an earlier method (Tsao, 1967) was used. Briefly, it involved growing the fungus in 25 ml V8-CaCOJ broth in a 12-0~ prescription bottle for 7 days after which 100 ml of water was added, causing the my&al mat to submerge under a liquid of 50-mm depth. The culture was then incubated for 2-3 weeks at 18°C in the dark. The washed, spore-bearing mycelial mat was blended, centrifuged briefly (15 set) twice to segregate the heavier chlamydospores from the mycelial fragments, and the chlamydospores centrifuge-washed three times before use. Oospores of P. megasperma var. sojae were produced by the method of Partridge and Erwin (1969). Harvested oospores were provided by D. C. Erwin. Methods of labeling

Fungal propagules were labeled by either of two methods : (a) direct staining of harvested fungal propagules, and (b) pre-labeling of fungal mycelium by growing the fungus in the brightener-containing liquid medium. Direct staining was made by incubating washed propagules to be labeled in 100 ppm or 280 ppm aqueous solution of the brightener for 2-5 hr in the dark. The spores were washed by centrifugation three times before use. Spores of some fungi germinate quickly in the absence of nutrients and a small percentage of these spores would have germinated at the end of the labeling period, making the labeling method undesirable. Therefore, to prevent germination during labeling, a 0.1~ phosphate buffer (pH 7) was used. This buffer was fungistatic to germination, and can be used to replace water in the labeling medium. These spores after washing retained their full germinability. Pre-labeling the fungal mycelium prior to spore production was used in most of the studies. It involved growing the fungus in the V8-CaC09 broth medium containing various concentrations of the brightener. M2R at a fmal concentration of 280 or 300 ppm was used in most experiments involving P. parasitica. A 5- to 7-day-old my&al mat grown in the labeled medium was centrifuge-washed three times to remove the nutrients and brightener in the medium prior to inducing the production of sporangia, zoospores or chlamydospores. These reproductive propagules were as highly fluorescent as the labeled mycelium from which the spores formed. Following harvest and washing, these propagules could be used immediately. Soil smear technique for recovery of propagules from soil

Saprophytic behavior of P. parasitica and other species in soil was studied by introducing labeled propagules of the fungus directly into the soil and recovering them periodically for observations. In studying soil fungistasis in relation to spore germination or in following morphogenesis of germinated spores in soil, spore suspensions were added directly to soil. A predetermined volume of a spore suspension was pipetted evenly to a Zmm-deep layer of known amounts of soil in small (60 mm dia.) Petri plates, reaching a moisture level of 24-32 per cent by weight (depending on soil types and the nature of experiments). After incubation at desired temperatures for varying lengths of time, soil samples were taken from

250

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the plates and soil-smear slides were made. About 100 mg of the infested soil (three soil chunks, each about 3 mm cubic in size, taken from three randomly selected sites) was placed on a 3 x 1 in. microscope slide, a drop (O-05 ml) of water added, large soil particles and sand grains carefully removed, and the soil smeared out in a thin layer with the aid of a halfspear needle. After a slight drying (about 4-5 min) at room temperature, S-10 drops of immersion oil of low degree of fluorescence were added as a mountant on which a 22 x 40 mm cover slip was placed. The slides were then ready for examination under a fluorescence microscope. In practice, however, slides were stored at 1°C in the dark until examination. Fiber glass technique *for recovery of propagules from soil In studying mycelial growth, sporulation or lysis, a fiber glass technique similar to that of Legge (1953) was used as an additional method for introducing fungal propagules into the soil and later recovering from it. Sterilized, 2 x 2 cm fiber glass squares were placed on a suitable agar medium and 2-3 drops of a suspension of the labeled spores were added to each square. Following spore germination and hyphal growth, the fiber glass squares with the enmeshed germ tubes or mycelium were carefully stripped from the agar surface and rinsed in three changes of water before incorporation into soil in either vertical or horizontal position. After desired lengths of exposure in soil, each fiber glass square was carefully retrieved, briefly dipped in water to dislodge large soil masses, and mounted in immersion oil as described for the soil smear technique. Fluorescence microscopy The labeled propagules and the hyphae and spores subsequently produced in the soil fluoresced a bright bluish-white when viewed with a Leitz Ortholux microscope equipped with an Osram HBO-200 mercury vapor lamp, a UGl excitation filter (with a transmission maximum in the 366 rnp region), and a U.V.absorbing barrier filter. Photographs were made on Ektachrome (ASA 160) or Panatomic X (ASA 32) fiIms. WEWLTS

E#ects of the brighteners on mycelial growth, sporulation and spore germination

Preliminary experiments testing the brighteners M2R, ST and PMS, each at concentrations of IO, 100, 1000and 10,000 ppm in cornmeal agar medium (Difco, 17 g/l), showed that all three compounds were inhibitory to mycelial growth of P. parasitica at a concentration of 1000 ppm or above, but were non-inhibitory or only slightly inhibitory at 100 ppm or below. At 100 ppm, the brightener M2R induced a ffuorescence in the mycehum more brilliant than either the ST or PMS formulation. M2R was, therefore, chosen in subsequent labeling experiments employing the V%CaCO, broth medium. M2R below 300 ppm in the broth medium generally produced no inhibition of mycelial growth of P. parasitica. Chlamydospores of P. parasitica were chosen as the propagule to study the effect of M2R on sporulation. Concentrations of 0, 140, 280 and 420 ppm were evaluated in two experiments in which M2R did not affect the ability of the labeled mycelium to produce chlamydospores (Table 1). M2R at 280 ppm also did not affect the ability of the washed, labeled mycelial mats of P. par~itica to produce sporan~a subsequen~y in water. The numbers of sporangia produced on the nonlabeled and labeled mycelial mats were similar in many experiments. The zoospore suspensions produced from such sporangia were also similar in concentration.

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(h52R) ONcHLAMyD(xIpoRHPRODUCTION TABLE~.EFFECTOFTHEBRK3~ BY Phytophthora parasitica

Expt.

No. of chlamydospores/lOO x microscope field+ Culture age (weeks) 1 2 3 4

Brightener Concn (ppm)

1

2

0

-

-

366

387

;: 420

-

-

366 -

360 376 380

160 172

385 356

408 417

0 280

3 1

*Average of 3 replicate bottles per treatment; 20 random fields per bottle culture. Less than 2 per cent of the spores counted were sporangia. Diierences were not statistically sign&ant (at both O-01 and 0.05 levels) between any treatments at each weekly interval.

Experiments were conducted on the effect of M2R on the germinability of labeled chlamydospores. Chlamydospores of P. pamsitica do not germinate in deionixed water (Isa0 and Bricker, 1968), but germinate well in a simple glucose-asparagine (each at 0-01~) broth medium. M2R at 280 ppm did not affect (either stimulate or inhibit) the germination of P. parasitica chlamydospores in deionixed water or in nutrient media (Table 2). TAB= 2. GERkWAnoN OF

Phytophthoraparastt&am

LARRLEDORLARRLED

Expt. 1

2

Chlamydospore Type+

THAT WERE NON-

WITHTHEBRI~~TENER

‘A Viibilityf

Nonlabeled Labeled

89 89

Nonlabeled Labeled

92

h42R

% Gerrninati;udz Deionixed water asparagine broth

0 0

100

i-3

100

86

spores% Cornmeal aear

96

98 94

99

-

+Nonlabeled and labeled chlamydospores were obtained from cultures grown in V8-C.aCoS broth containing 0 and 280 ppm, respectively, of the brightener. tNonviable spores were stained red by a 60 ppm aqueous rose bengal solution. $Germination was read at 16-18 hr at 25°C. Figures were based on counts of 100 chlamydospores per replicate, 3 replicates per treatment. Differences in percent germination between the nonlabeled and labeled spores were not statistically sign&ant (at both 0.01 and O-05 levels).

In another experiment, the effect of M2R on the subsequent germinability of several additional spore types of five fungi were tested. Direct staining technique was used and the concentration of M2R tested was 280 ppm. Except for oospores of Phytophthora megasperm var. sojae, there was no difference, in the degree of germination, between the nonlabeled and labeled spores of any other fungi tested (Table 3).

2.52

P. H. TSAO TABLE 3. GERMINATION OF VARIOUS FUNGAL SPORES THAT WERE NONLABELED OR LABELED BY DIRECT STAINING FOR 2 hr WITH 280 ppm OF THE BRIGHTENER M2R

FURgUS

Fusarium solani ThieIaviopsis basicola ~erti~~Ii~~rnaIbo-atrum Phytophthora megaspermd var. sojae Phytophthora parasitica

Spore form

Macroconidia Microconidia Endoconidia Chiamydospores Conidia oospores zoospores Chlamydospores (as control)

“/, Ge~i~ation* Noniaheied k&led SjKWCS spores 100

100

100 96.3 92-7 99

99.3

24.1 85

37.1 90.7

77.3

76.3

g.3 99‘3

*For all spores, except the oospores, the ration tests were made at 25°C on potato dextrose yeast extract agar, and -nation was terminated at 16-18 hr. For the oospores of P. megasperma var. sojae, the germination test was made in water and germination terminated at 96 hr. Figures were based on counts of 100 spores per replicate, 3 replicates per treatment. Except with the oospores. differences irt percent germination between the nonlaheIed and laheled spores were not statistically significant (at both 0.01 and O-05 levels). Fluorescence of labeled fungal propaguies in vitro

Mycelium of P. parasitica and the other 11 Phytophthora spp. tested (see ‘Materials and Methods’) fluoresced brightly immediately after labeling with 100 ppm or higher concentrations of the brighteners M2R, ST or PMS. Mycelia of F. solani and Y. aibo-atrum fluoresced to a lesser degree. Mycelium of T. b~i~ola fluoresced only dimly, but the septa showed a brighter fluorescence. Encysted zoospores of P. parasitica fluoresced brightly when labeled with the brighteners M2R, ST or PMS, each at 100 ppm. Encysted zoospores of the other 11 Phytophthora spp., when labeled with 100 ppm M2R, also fluoresced to the same degree as P. parasitica zoospores. Motile zoospores of these Phytophthora spp., however, did not fluoresce. The phenomenon is reported in detail elsewhere (Tsao, 1969b). When the brightener RW was used, motile zoospores of all species fluoresced. Sporangia of all the Phytophthoru spp. tested fluoresced when labeled with any of the brighteners used. MtR-labeled chlamydospores of P. parasitica and oospores of P. megasperma var. sojae both fluoresced brightly. Fluorescence of the labeled spores of other fungi tested varied greatly. When Iabeled for 2 hr with 280 ppm M2R, both macroconidia and microconidia of F. solani fluoresced brightly, especially the septa of the macroconidia. Conidia of V. a~bo-ate were similar to F. solani in the degree of brightness. Fluorescence of endoconidia of T. basicola was discernible but moderate to dim. The dark pigmented chlamydospores of T. basicoia labeled with 280 ppm M2R, however, did not fluoresce. Other pigmented, melanized fungal structures such as the microsclerotia of VerticilZium are also known to show no fluorescence whereas the hyaline mycelium and conidia are fluorescent (C. E. Horner, personal communication). Spores of the fungi listed in Table 3 were incubated for 16 hr in VS-CaC03 broth containing 280 ppm M2R to allow germination after which fluorescence of germ tubes was compared. Germ tubes of F. solani macro- and microconidia ffuoresced brightly. Although

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chlamydospores themselves were nonfluorescent, germ tubes of both endoconidia and chlamydospores of T. basicola fluoresced, but dimly. Fluorescence was much brighter at the septa of these germ tubes. Fluorescence of germ tubes of ‘I/ albo-atrunzconidia was also dim. Both zoospores and chlamydospores of P. parasitica produced brightly fluorescent germ tubes. Germ tubes from labeled oospores of P. megasperma var. so&e, however, produced only a dim fluorescence. When fungal propagules were labeled with the brightener at concentrations between 100 and 300 ppm, they all exhibited about the same degree of fluorescence immediately after labeling. The degree of ffuorescence of the sub~quently formed vegetative or reproductive structures from the labeled propagule, however, varied and appeared to be dependent on the concentration of the brightener and the length of the labeling period. Flourescence of labeled fwgal propagules in soil

M2R-labeled mycelium, sporangia, zoospores and chlamydospores of P. parasitica fluoresced brightly when incorporated into natural soils and viewed on soil-smear slides. The fungal structures subsequently formed in soil from the labeled propagules also fluoresced and could be easily distinguished with little or no difference from soil particles and other soil microflora (Fig. I). The adhering soil particles on fungal hyphae or germ tubes, which greatly obscured the visibili~ of these fagal structures under white illu~nation, were unnoticeable under U.V.illumination. Furthermore, with the fluorescent labeling technique, only the labeled test fungus added to the soil, but not other microflora, was detected on the soil smear slides. Labeled fungal propagules, especially large spores, contained amounts of the brightener suScient to be transported to newly formed vegetative or reproductive structures for a considerable distance and duration. For example, a labeled mycelial mat of P. parasitica after incorporation into a moist natural soil produced in situ large numbers of chlamydospores and sporangia, both brightly fluorescent when recovered and viewed on soil-smear slides. Labeled c~~ydos~~s, produced in oitro and added to natural soils, usually germinated by producing a short germ tube with a terminal sporangium (Fig. 1 C; 2 A, B). These fiuorescent sporangia often germinated in soil by releasing zoospores which were also fluorescent. When these zoospores encysted and germinated, fluorescent germ tubes (Fig. 2 C) with only a slight diminution in the degree of fluorescence were observed. Thus, saprophytic behavior and morphogenetic changes of various fungal propagules can be traced directly in soil despite the background opacity. Applicationsto ecological studies of Phytophthora parasitica in soil

With the vital fluorescent labeling technique, various saprophytic activities of several factors have been successfully studied. Most of the work dealt with spore germination, myoelial growth, sport&&on and lysis of P. parasitica in non-amended and amended natural soils. This paper is not meant to present the results in detail, but only to report the applications of the labeling technique to such ecological studies. Germination of labeled chlamydospores and zoospores of P. parasitica on soil-smear slides is shown in Fig. 2 (A-E). Unlike spores of many other fungi (Lockwood, 1964), chlamydospores of P. parasitica germinated well in moist non-amended natural soil; germination often reached above 70 per cent. A slightly higher germination was observed in autoclaved soils or soils amended with various organic substrates. lenient with amino acids or ammonia nitrate inhibited chlamydos~~ germination in soil, however

P~ytop~t~~a spp. in natural soils as in~uenced by soil enviro~ent

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P. H. TSAO

(Tsao and Bricker, 1968; Tsao, 1969c). Zoospores also germinated in nonamended soil, but at lower percentages than chlamydospores. Occasionally, unusual behavior of germ tubes was observed, involving the formation of appressorium-like structures (Fig. 2 D, E) and microsporangia (Fig. 2 E). Mycelial growth ofP. parasitica in nonamended and amended soils has also been studied using the vital fluorescent labeling technique. Average areas of fluorescent mycelial growth, on soil-smear slides, ranged from less than 100~ to more than 1000,~ in diameter (Tsao, 1969c). P. parasitica grew poorly or not at all in nonamended soils, but grew in measurable degree when soils were amended with 0.4-2 per cent of D-glucose or L-asparagine. The extent of mycelial growth varied in different soils and was greatest during the first two days after amendment in natural soils. Mycelium continued to grow, though slowly, for several days in amended autoclaved soil. The results are reported in detail elsewhere (Tsao, 1969c). The use of vital fluorescent Iabeling for the study of in situ sporulation of P. parasitica in natural soil was attempted. Chlamydospores were formed on freshly growing mycefia in amended natural soils (Fig, 1 D; 2 F). The germ tubes newly formed from the chlamydospores generally produced sporangia if soils were nonamended (Fig. 1 C; 2 A, B). Amending the soil with glucose enhanced the growth of germ tubes to form extensive mycelium, and prevented or retarded sporangium production. Periodic samplings of infested soils employing soil-smear slides revealed that, in a glucose-amended natural soil, the number of sporangia and c~amydospores increased as the amount of fluorescent myce~um area in soil-smear slides decreased. Decrease in the amount of fluorescent mycelium is construed to be the result of lysis of the mycelium. With most of the natural soils studied, lysis generally became evident at 4 days. Very little mycelium was observed at 14 days. In autoclaved soils, lysis was negligible even at 14 days. Some of the results on sporulation and lysis have appeared previously (Tsao, 1969c). The fiber glass recovery technique (Fig. 2 G) proved adequate as a supplementary technique to the soil smear method. In non~ended natural soil, there was little or no difference between the average lengths of germ tubes in the fiber glass squares at 0 day and at 1 or 2 days. In glucose-amended soil, germ tubes increased in length, branched, and formed extensive mycelium. Sporulation and lysis data obtained with the fiber glass technique were similar to that of the soil smear technique but were less quantitative. Viability of the propagules recovered in duplicate fiber glass squares can be tested by plating the squares on a selective antibiotic medium (Tsao and Ocana, 1969). Autofluorescence of the fiber glass threads and poor recovery of fungal propagules are two undesirable features of the fiber glass technique, making it less useful than the soil smear technique. DISCUSSION

The fluorescent brighteners satisfy the requirements for an effective vital labeling substance that can be used to study the ecology of Phytophrhora and certain other fungi in soil. At the concentrations used, M2R is essentially noninhibitory to growth, sporulation and spore germination of P. parasitica and several other fungi tested. It is readily absorbed and transported to subsequently formed vegetative and reproductive structures. When labeled at sufficient concentrations, the fungus fluoresces intensely, even in subsequently developed structures. M2R apparently has an affinity for fungal cell wall components (Tsao, 196915). The binding of the brightener to the fungus is highly stable; the labeled fungal propagules on soil-smear slides showed no detectable loss of fluorescence even after 36 months of storage at 1“C in the dark. The soil smear technique used in this study for the recovery of fungal propagules differs

FIG. 1. Brightener-labeled propagules of Phytoph?hora parusiticu in soil-smear slides as viewed under white illumination (A, B) and U.V.illumination in fluorescence microscopy (C, D). Spores and hyphae fluoresce brightly under U.V.and C&Ibe de&ted with little interference from soil particles and other soil microflora. In the A-C pair, a chlamydospore which germinated within 24 hr in a nonamended natural soil, producing a germ tube with a terminating sporangium. In the B-D pair, growth of new hyphae with two intenzalary chlamydospoms, in a glucoseamended natural soil 2 days after soil infestation with labeled chlamydospores.

SBB f.p. 2541

FIG. 2. Saprophytic behavior of P&tophrhora parusitico in soil as studied by the use of brightener-labeled fungal propagules and fluorescence microscopy. A-F-with soil-smear technique; G-with fiber glass technique. A-A chlamydospore germinated within 24 hr in a nonamended natural soil, producing a germ tube with a terminating sporangium. B-Same as A, except in a different soil. C-Germinated zoospores which had been released from the sporangia produced on the germinated chlamydospores pre-labeled and used as the inoculum in soil infestation. Althoughchiamydospores were the initially iabeied propagules, germ tubes from the subsequently produced zoospores in the l-day-recovery soil-smear slide were still fluorescent. D-A zoospore germinated in a nonamended sterilized soil, producing a series of appressorium-like structures. Recovery by soil smear was made 2 days after soil infestation with labeled zoospores. E-Same as D, except recovery was made at 4 days. The germ tube of the zoospore at the bottom has a terminating ‘microsporangium.’ F-Growth of new hyphae, with subsequent chlamydospore production. in a glucose amended natural soil 24 hr after soil infestation with labeled chlamydospores. G-Germinated zoospores enmeshed in a fiber-glass square prior to incorporation into the soil.

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somewhat from those employed by other workers. It involves larger amounts of soil and the use of immersion oil as the moun~nt. Also, no color dyes are needed to stain the fungus. The direct soil infestation methods accompanied by any conventional soil smear or other recovery techniques have received the universal criticism that extremely high concentrations of fungal inoculum are needed for an adequate later retrieval of the fungus. The inoculum concentrations used in these methods often far exceed those existing in natural soil conditions. Due to the freedom from interference by soil particles, the present technique permits the sampling of a large amount (e.g. 100 mg) of soil on each slide and, hence, a concomitant use of a more realistic spore concentration (e.g. 10,000 spores/g) for initial soil infestation. The vital fluorescent labeling technique is superior to some other direct methods especially in the study of spore germination in soil. With most of the direct methods devised for studies on soil fungistasis, germinated spores with their long, stained germ tubes are easy to locate, but nongerminated spores, especially those of small sizes, are extremely difficult to differentiate from small, nonspore particles. When a portion of the nongerminated spores are not recognized, the percent germination figure tends to be higher than it should be. The brighteners render fluorescence only to the introduced fungal propagules. 3oth nongerminated and germinated spores in the soil smear slides are equally detectable against a nonfluorescent soil background. The conventional color dyes used in all direct methods for studying fungal behavior in soil indiscriminately stain all stainable microflora. In soils amended with various organic amen~en~ most fungi, in addition to the test fungus and other microflora, flourish to a degree which often interferes with the detection of the test fungus, since mycelia of different fungi are generally not morphologically distinguishable. The vital fluorescent labeling technique reliably distinguishes the test organism from other soil microorganisms. Another versatile and related technique widely used for studying microbial activities in soil is the i~uno~uore~nt staining technique (Eren and Pramer, 1966; Hill and Gray, 1967; Schmidt et al., 1968). Although viewing is accomplished by fluorescence microscopy in both cases, the two techniques differ in principle, in application and in usefulness. The immunofluorescent staining technique involves an elaborate antibody production procedure and is useful for the detection and identification of the organism in question mainly in its native state. The vital fluorescent brightener labeling technique, on the other hand, involves only a simple labeling of the test organism in vitro and is used for tracing the develop ment of the labeled organism after it has been introduced into the soil. The brightener labeling technique is not without disadvantages. Like all techniques involving soil smears (Nash et al., 1961) or soil suspension plating (papavizas, 1967), the natural positions of fungal spores, germ tubes, and hyphal branching, and their spatial relationships in soil are often disturbed. The distortion can be minimixed if the smearing is done gently and rapidly before the water in the soil slurry begins to evaporate. A second drawback is that certain organic substances naturally present in some soils exhibit a low degree of primary fluorescence, as do some fungi and some of the organic. amendment used, e.g. alfalfa meal. Also, if not adequately labeled, fungal propagules eventually lose fluorescence due to the dilution of the brightener in a rapidly growing fungus. Despite these shortcomings and inconveniences, the vital fluorescent labeling technique is an extremely versatile and effective method for studying saprophytic behavior and activities of various ~~oorganisms in soil. Since the initial application of brighteners to ~~obiolo~cal studies nine years ago (Darken, 1961), few attempts have been made to adapt it to studies in soil microbiology (Eren and Pramer, 1968; Tsao, 1969a). It is hoped that this report will stimulate further studies in this area,

256

P. H. TSAO

Ackno~ledgmeats-~ thank J. L. BRICKERfor able technical assistance; B. T. HAWIXORNE for his suggestion on the possibb application of brighteners to soil ecoIogica1 studies and for his assistance in the intial in vitro experiments; D. C. ERWM for providing cultures of VerticiiIium aibo-atram and Phytophthora megaspermu var. sojae; D. W. CHENand G. A. ZENTMYERfor providing sporangial cultures of many Phytophthora spp. used in this study; W. H. MCCORMICK for obtaining oospore germination data; and American Cyanamid Company for providing the brighteners.

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