Physiology, chemistry, serology, and infectivity of two Frankia isolates from Alnus incana subsp. rugosa

Physiology, chemistry, serology, and infectivity of two Frankia isolates from Alnus incana subsp. rugosa

MARY P. LECHEVALIER Waksrnan Institute of Microbiology, Rrctgers University, P.O. Box 759, Piscataway, N J , U.S.A. 08854-0759

DWIGHT BAKER' Department of Biology, Middlebury College, Micldlebury, VT, U.S.A. 05753

AND

FRANCOISE HORRI&RE?- Waksrnan Institute of Microbiology, Rictgers Ur~iversity, P.O. Box 759, Piscataway, N J , U.S.A. 08854-0759

Received December 1, 1982

LECHEVALIER, M. P., D. BAKER, and F. HORRI~RE. 1983. Physiology, chemistry, serology, and infectivity of two Frartkia isolates from Alnus incana subsp. rrcgosa. Can. J. Bot. 61: 2826-2833.

Actinorhizal nodules of Alnus incarm ssp. rugosa collected from plants growing within 30 km of each other in Jeffersonville and Stowe, VT, U.S.A., yielded two morphologically distinct strains of Frar~kia. A comparison of the cell chemistry of the strains showed that each had the same type of cell wall and whole-cell sugar and phospholipid composition; however, Air12 lacked the unknown amino acid found in the cells of all frankiae previously examined. Their cellular fatty acid composition showed only minor qualitative differences. Physiologically, the strains differed in their relation to oxygen, in their uptake and utilization of carbohydrates, organic acids, and lipids, and in protease production. AirIl induced effective nodules in the host plant; those of Air12 were ineffective. Air12 was unrelated serologically to all frankiae previously isolated from the genus Alnus. AirIl was serologically typical.

LECHEVALIER, M. P., D. BAKER et F. HORRI~RE. 1983. Physiology, chemistry, serology, and infectivity of two Frankia isolates from Alnus incana subsp. rugosa. Can. J. Bot. 61: 2826-2833.

Des nodules actinorhiziens d'Alnus incana ssp. rrcgosa rCcoltis de plantes poussant a quelque 30 km les unes des autres, B Jeffersonville et ?i Stowe dans le Vermont (U.S.A.), furent isolCes deux souches de Frankia morphologiquement nettement diffkrentes l'une de l'autre. Ces deux souches avaient le m&me type de paroi cellulaire et la m&me composition en sucres cellulaires et en phospholipides; par contre, l'une d'elles, AirI2, ne contenait pas l'acide aminC non-identifiC qui avait CtC trouvi jusqu'ici dans les cellules de toutes les souches de Frankia. On observa seulement de petites diffkrences qualitatives dans les compositions en acides gras de ces deux souches. Physiologiquement, les deux souches diffiraient quant a leurs besoins en oxygkne, leur absorption et utilisation des hydrates de carbone, des acides organiques et des lipides et aussi dans leur production de protiase. Les deux souches avaient la capaciti de produire des nodules sur les racines d'aulnes mais celles de Air11 Ctaient effectives tandis que celles de Air12 Ctaient ineffectives. La souche AirIl etait sirologiquement sernblable a toutes celles qui furent isolCes jusqu'ici d'aulnes tandis que Air12 ne pouvait &tre placCe dans aucun des groupes serologiques reconnus jusqu'ici pour les souches de Frankia.

Introduction Many actinomycetes belonging to the genus Frankia

have now been isolated from a variety of host plants (Baker 1982). To our knowledge, all isolates from the same host have been very similar; thus the report of the isolation of two morphologically distinct strains of Frankia from nodules of the speckled alder, Alnus incana ssp. rugosa (Homkre et al. 1983), appears to be unique. This paper describes a comparison of the cell chemistry, physiology, serology, and infectivity of these two different strains, AirIl and AirI2.

Materials and methods Isolation of cultures

Nodules of Alnus incana ssp. rugosa were collected in

'present address: Charles F. Kettering Research Laboratory, 150 Eastsouth College St., Yellow Springs, OH, U.S.A. 45387.

'present address: 2 impasse Fouillonaz, St. Romain-au- Mont d'Or, 69270 Fontaines-sur-SaBne, France.

Stowe and Jeffersonville, VT, U.S.A., and were worked up immediately without freezing. Both AirIl and Air12 were isolated by incorporation of NaOC1-sterilized, crushed nodules into water agar and 1116-strength Bennett's agar (Higgins et al. 1967) containing actidione at 500 p,g/mL and nystatin at 30 p,g/mL. The plates were incubated at 28OC for 2-4 months until colonies appeared.

Media After isolation, AirIl was maintained in L /2 medium

(Lechevalier et al. 1982) and Air12 on yeast - Czapek's (YCz) agar (Higgins and Lechevalier 1969) or 0.5-strength Bennett's broth. Basal medium "S" used for studies of glucose-Tween interactions contained casein hydrolysate (NZ Amine A, Sheffield Products, Memphis, TN), 4.0 g; K2HP04, 500mg; MgS04.7H20, 200 mg; CaC12.2H20, 100 mg; minor salts and fenic citrate of Qrnod medium (Lalonde and Calvert 1979); distilled water, 1 L; pH 6.8 before autoclaving. Tween 80 (poly- oxyethylene sorbitan monooleate) when added was at 0.2%. Organic acid decarboxylation was determined in a basal medium containing NZ Amine A, 5.0 g; NaCl, 1.0 g; MgS04.7H20,

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0.2g; KH2P04, 0.5 g; FeNaEDTA, 0.01 g; CaCI2,2H20, 0.01 g; 0.04% phenol red, 20 mL; distilled water, 1 L; pH 6.8. Organic acid salts were added to the medium at 0.2% prior to autoclaving. Decarboxylation leads to a rise in pH assessed as a change in color of the indicator from yellow-orange to red. Nitrate utilization was determined in Czapek's medium; nitrate as an electron acceptor in nitrate agar (Difco beef extract, 3 g; Bacto peptone, 5 g; KN03, 1 g; Bacto agar, 12 g; distilled water, 1 L; pH 7.2; 12 mL per 16 x 150 mm tube); and ammonium or glutamate utilization in Koser's medium: (NH4)2HP04 (L-glutamic acid, 1.5 g), 1.0 g; glucose, 10 g; KC1, 0.2 g; MgS04.7H20, 0.2 g; distilled water, 1 L; pH 6.6 before autoclaving. Other physiological test media have been described previously (Lechevalier et al . 1982).

Cell chemistry Whole-cell, cell wall, and phospholipid analyses were

performed according to Lechevalier and Lechevalier (1980). Saponification of neutral lipids was carried out in 1.3% methanolic potassium hydroxide under reflux for 2 h. After extraction with petroleum ether (bp 40-50°C) (PE), the reaction mixture was adjusted to pH 1.0 with 6 N HC1 and reextracted with PE three times. The second PE extract, taken to dryness under nitrogen, was methylated with 10% boron- trichloride-methanol according to Lechevalier and Lecheval- ier (1980). Neutral lipids were analyzed and purified on Brinkmann PF254 G60 silica-gel plates 0.5 mm thick, de- veloped in PE:ether: glacial acetic acid, 80:20:2, and sprayed with 0.1 % ethanolic Rhodamine B : 0.25 M KH2P04 (1 :9). Methyl esters of fatty acids were analyzed by gas chromatography as described in Lechevalier et al . (1977).

Plant studies Infectivity trials were performed using the following plants

as potential hosts: Ainus incana ssp. rugosa, A . rubra, A . viridis ssp. crispa, Ceanothus americanus, and Elaeagnus umbellata. Inoculation procedures were as described in Baker et al . (1980). The acetylene-reduction test as described by Dillon and Baker (1983) was employed to determine nitrogenase activity.

Serology Serological analysis of the bacterial strains was performed

using a double-diffusion immunodiffusion procedure as described by Baker er al . (1981).

Scanning electron microscopy (SEM) Whole nodules were fixed in 2% buffered glutaraldehyde for

2 weeks to 1 month at room temperature or 3 h at 4"C, then prepared according to Baker et al. (1980) and observed on either a AMR 1000 or Joel JSM 35C.

Results Morphology

Frankia sp. Air11 is very similar to all other frankiae in our collections which have been isolated from the genus Alnus. This includes isolates from A. rubra, A. viridis ssp. crispa, A. viridis ssp. sinuata, and A. glutinosa. Like them, it forms an essentially colorless to white growth on a variety of different media. The branching hyphae, 0.8-1.5 km in diameter, bear ter- minal, lateral, and intercalary sporangia containing

spores concolorous with the hyphae, AirI2, in contrast, forms narrower light-tan to orange-tan hyphae measuring 0.5-0.75 km with sporangia containing black spores measuring 0.5-0.7 km. On slants, the growth of Air12 is at first colorless, then tan-orange, and finally black as the spores within the sporangia mature. Neither strain forms vesicles on ordinary culture media.

Cell chemistry Chemical analysis of the cells of Frankia spp. AirIl

and Air12 showed them to be like all the other frankiae we have analyzed to date, having a cell wall composition of type I11 (meso-diaminopimelic acid, glutamic acid, alanine, muramic acid, and glucosamine as principal components of the peptidoglycan). Air12 differs from them and Airll, however, in its lack of the polar, blue-staining unknown amino acid we have reported from other Frankia strains (Lechevalier and Lechevalier 1979). Also like all other frankiae from Alnus spp. (Lechevalier and Lechevalier 1979; Baker et al. 1981) both strains had a whole-cell sugar pattern of type D (xylose and arabinose). In addition each contained substantial amounts of the unknown hexose (Lecheval- ier and Lechevalier 1979) which may be identical with that found in Frankia sp. CpIl and identified as 2-0-methyl mannose (M. Lalonde, cited in Wheeler 1981). The phospholipid patterns of the two strains were like those of other frankiae, namely, of type P I (Lechevalier and Lechevalier 1980). This type is charac- terized by a content of phosphatidyl inositol, phosphatidyl inositol mannosides, and diphosphatidyl glycerol. Nitrogen-containing phospholipids are lacking.

Physiology AirIl and Air12 differ in their physiology. AirI1, like

previous isolates from Alnus nodules, is strictly microaerophilic and cannot be maintained in slant culture. AirI2, on the contrary, has been maintained since its isolation (3 years) on YCz slants (Higgins and Lecheval- ier 1969). Both strains grow in a temperature range of 10-33°C; little to no growth occurs at 37°C. In addition, there are pronounced differences in their utilization of lipids and carbohydrates. Typical growth curves for both strains in basal medium "S" with or without the addition of 0.2% Tween 80 and with various concentrations of glucose are given in Fig. 9.

In this medium, Tween 80 is utilized as a sole source of carbon by AirIl but not by AirI2. Glucose alone, without Tween, is utilized better at a 1% concentration by Air12 than AirI1; however, at 2%, Air11 produces the greater cell mass and this trend is continued at 3% (data not shown). If 2% glucose is added to the Tween- containing medium, a synergistic effect with the Tween on cell growth is apparent in AirI1, the resultant cell weights being 66% greater than the sum of the cell weights from media containing each of these substrates separately. In contrast, in AirI2, addition of Tween has

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the effect of depressing growth as much as 50%. The reason for this toxicity becomes apparent if one observes the cells of the strain grown in such a medium with a microscope: large globules of free fatty acids are intermingled with the hyphae. Thin-layer chromatography and gas-liquid chromatography (GLC), compar- ing the neutral lipids of AirIl and Air12 cells grown in S medium plus Tween and 2.0% glucose, show that whereas the fatty acids (oleate) released from the Tween 80 are incorporated into the cellular lipids of AirI1, they remain largely free in AirI2. As free fatty acids are known to be toxic, this is undoubtedly the cause of the depressed growth of the latter.

GLC analysis of the fatty acids released by saponification of the total cellular lipids of late stationary-phase cells of AirIl and Air12 grown in S medium without addition of Tween or other exogenous source of fatty acids shows that the strains are quite similar. As can be seen from Table 1, the major fatty acids were iso- hexadecanoic, n-hexadecanoic, and n-octadecenoic. Fatty acid analysis of the purified triglyceride fraction of AirIl grown in S medium plus 0.2% Tween 80 is included for comparison and shows the incorporation of oleate into the cellular lipids of this strain. There is also a marked diminution of iso-hexadecanoic and the appear- ance of new unsaturated fatty acids, including hexa- decenoic and heptadecenoic acids.

Nitrate cannot be utilized as a source of N or as an electron acceptor for anaerobic growth by either strain; nor can it be cometabolized to nitrite in the presence of amino acids. Ammonia and amino acids are utilized by both strains.

As little to no carbohydrate is taken up by Air11 at a concentration of 0.5%, but is by AirI2, one can differentiate between the strains on this and other bases such as production of protease (caseinase) and capacity to decarboxylate certain organic acids (Table 2).

AirIl and Air12 are representatives of two distinct groups of frankiae (Table 3) which may be discerned among the strains presently held in our collections. The first group, which we have termed type A (Lechevalier and Lechevalier 1983), contains strains which utilize and produce acid from a variety of carbohydrates at a concentration of 0.5%. They usually produce protease (caseinase) and have pigmented cells. They tend to be more aerobic than the second group (B) and can be maintained on slants. Their whole-cell sugar patterns are very diverse, they are ineffective in the host plant, and they do not fall into the serological group (serotype I) characteristic of the strains in group B. Group A includes, besides AirI2, Frankia spp. G2 (Gauthier et al. 1981a, 1981b), EuIlb (Baker et al. 1980), PtIl (Baker 1982), and CaIl (F. Horriere and M. P. Lechevalier, unpublished data).

AirIl is typical of type-B strains. Most of these do not utilize carbohydrates at 0.5%, produce colorless

TABLE 1. Fatty acid composition (percent of total) of cellular lipids of AirI 1 and Air12

Air1 1 Air12 -

Fatty acid CFA TF A CFA

iso-C- 14 n-C-14 n-C-15 Unknown iso-C- 16 C-16'= n-C- 16 Atzteiso-C- 17 C-17'' n-C-17 Unknown Anteiso-C- 18 C-18"

NOTE: CFA, total cellular fatty acids, cells grown in S medium with 2.0% glucose; TFA, fatty acids from purified triglyceride fraction, cells grown on S medium plus 2.0% glucose and 0.210 Tween 80; '=, monounsaturated fatty acid.

TABLE 2. Comparison of utilization of various carbon and nitrogen sources by Frankia spp. AirIl and Air12

AirIl Air12

Nitrogen sources Amino acids (e.g., casein hydrolysate) Nitrate Ammonia Glutamate

Carbon source" Cellulose Arabinose Glucose Glycerol Maltose Sorbitol Sucrose Xylose

Decarboxylate Acetate Propionate Pyruvate Succinate

Degrade Casein Starch

"In NZ, medium (Lechevalier et al. 1982) with no added Tween

growth, are strictly microaerophilic, and do not produce proteases under the conditions used. All have a whole- cell sugar pattern of type D (xylose), form a tight group serologically, and are infective and effective in the plant host. Group B includes all the other Alnus isolates in our

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FIG. 1. Vesicles in field-collected nodules from same collection from which Air12 was isolated. X6160. Bar = 1.0 Km. FIG. 2. Vesicles of field-collected nodules; same collection from which Air11 was isolated. x3000. Bar = 1.0 Km. FIG. 3. Sporangia present in the nodules illustrated in Fig. 2. X 10 800. Bar = 1.0 Km. FIG. 4. Vesicles from effective nodules induced on Alnus rubra by AirI1. X 1150. Bar = 10 Km.

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FIG. 5. Hyphae of Air12 in ineffective nodules induced in A. incana ssp. rugosa. X2280. Bar = 1.0 km. FIG. 6. Hyphae of Air12 as in Fig. 5 showing formation of incipient sporangia(?). X2280. Bar = I .O km. FIG. 7. Vesicles in an effective nodule induced by Air11 on A. rubrn. X4200. Bar = 1.0 km. FIG. 8. Hyphae of Air12 lacking vesicles and sporangia in an ineffective nodule on A. incann ssp. rugosa. x2820. Bar = 1.0 km.

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TABLE 3. Physiology of some Frnnkin spp.

Carbohydrate source" Casein Starch

CultureNo. A G1 G2 M S I S2 X hydrolyzed hydrolyzedb Group'

EuIlb A , U A , U - U - - A, U + + D A (32 A , U U - A, U - U A, U +C A -

PtIl A , U A , U - U I U A , U + + D A Air12 - U - U - U A,- + + D A ArI3 - I - - - + D B - - - Air1 1 - 1 1 - - + D B - - -

AvsI2 - I - I + D B - - - - CpIl - + D B - - - I 1 I -

MpI 1 - - U - - U - - + D B?

"A, arabinose; G I , glucose; Gz, glycerol; M, maltose; S , , sorbitol; Sz, sucrose; X , xylose; -, not utilized; I , growth inhibited compared with basal medium; A, acid produced; U, utilized, growth increased over that of basal medium.

I + , hydrolyzed;-, not hydrolyzed; D, dextrinoid reaction with starch-iodine reagent; C, starch completely hydrolyzed, no reaction with starch-iodine.

'For explanation, see text.

collection and those from Comptonia peregrina, and Myrica pensylvanica, and M . cerifera.

Plant studies Nodulation by both strains of Frankia was observed

on all Alnus species but not on Ceanothus americanus or Elaeagnus umbellata. Nodules induced by strain AirIl typically appeared 7-14 days after inoculation and all plants inoculated with this strain became infected. In contrast, Alnus seedlings inoculated with strain Air12 became infected at a low frequency and nodules were visible only after 28-42 days postinoculation. Nodules induced by Air12 remained small and plants nodulated with this strain never recovered from nitrogen de- ficiency. Acetylene-reduction values for the Alnus spp. - AirI 1 symbioses were in the range of 7- 12 pmol C2H4 per hour per gram nodule fresh weight. These values are comparable with other symbiotic combinations reported previously (Dillon and Baker 1983). No detectable nitrogenase activity was recorded for plants nodulated by strain AirI2.

SEM's taken of the nodules induced by Air12 on

Time (weeks)

FIG. 9. Influence of Tween 80 and glucose on growth of Frankia spp. AirIl and Air12 in "S" medium. (A) Without Tween 80; (B) with Tween 80 at 2 mL/L. AirI1: 0, no glucose; A, 1 % glucose; ., 2% glucose. AirI2: 0, no glucose; A, 1% glucose; 0, 2% glucose.

Alnus incana ssp. rugosa in the greenhouse (Figs. 5, 6, and 8) show plant cells filled with hyphae but no indicated that strain AirIl was related to other strains

vesicles. This is consistent with the ineffectivity of the assigned to serotype I as defined by Baker et al. (1981)

strain. Some swellings which resemble the sporangia but that strain Air12 was not closely related to serotype I.

formed in vitro by this strain are also visible. In contrast, Air12 was more similar to strains assigned to serotype I1

in nodules formed by AirI1, both hyphae and vesicles as defined by these authors. Because Frankia strains

are observable (Figs. 4 and 7). An examination of the isolated from the same host species would be expected to belong to the same serogroup, the results observed with nodules from the original collections from which each

strain had been isolated (held at -20°C for 3.5 years) strains AirIl and Air12 indicate that the relationship

showed that vesicles were present in the nodules from between host compatibility and serotype is more com-

both localities (Figs. 1 and 2). Sporangia were also plex than originally interpreted (cf. Baker et al. 1981).

seen (Fig. 3) in nodules from the coilection that yielded Discussion AirI 1. Even prior to the first actual isolation and main- Serology tenance in pure culture of a true Frankia sp. (Callaham

Serologic analysis of these two Frankia strains et al. 1978), Quispel (1960) reported the enhancing

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effect of lipid-containing plant extracts on the infectivity of crushed nodules. This author has more recently reported that such lipids are obligatory for growth of both "spore-negative" and "spore-positive" frankiae from Alnus glutinosa (Quispel and Burggraaf 1981) and that these lipids could not be replaced by Tween 80. Since 1977, when one of us (M. P. Lechevalier) began handling frankiae in pure culture, media amended with Tween 80 have been used with good success for routine maintenance of many strains (Berry and Torrey 1979; Lechevalier et al. 1982). Blom and Harkink (1981) studied the effects of Tween 80 on the growth of Frankia sp. AvcIl and reported that the fatty acids released from Tween 80 could be used as a sole source of carbon but that glucose was not taken up by this organism after a 6-h contact at a concentration of 1 mM (0.018%), even in the presence of 0.2% Tween 80. As we have found that AvcIl is a Frankia of type B (M. P. Lechevalier, unpublished data), their findings are consistent with ours, namely, that at such low concentrations little to no glucose is taken up.

A possible explanation of the difference between the two groups of frankiae is that the A type has, and the B type lacks, an active carbohydrate transport system. At higher concentrations of glucose, the gradient across the cell envelope is increased and glucose can penetrate the "B" cell. The evidence for a Tween-glucose synergism clearly shows that Tween not only serves as a source of carbon but also as a "facilitator" of carbohydrate uptake. Whether this is a result of its amphipathic nature or whether the fatty acids released from it by the cellular lipases act to create passages in the outer and inner membranes or make them more "fluid" remains to be determined. It is known (Mindich 1973) that in other bacteria, lipids play a role in carbohydrate transport, but there is conflicting evidence on the exact nature of this association.

It seems likely that frankiae of the B type are regulated in planta by (i) glucose concentration in the nodules and (ii) amphipathic compounds of plant origin. Those of group A would tend to be more independent of the plant's photosynthetic variations.

Of interest also is the vesicular evidence that there are (presumably) nitrogen-fixing nodules present among those from which the Air12 strain was isolated. Thus it seems that either (i) the nodules at the Air12 site contain two types of endophytes, one effective and one ineffective or parasitic, (ii) the Air12 strain lost the capacity to form effective nodules during its isolation, or (iii) there is some critical factor missing when infection of the host plant is carried out in the greenhouse which prevents the strain from expressing its nitrogenase activity. Support- ing this last theory, Gauthier et al. (1981a, 1981 b) found that Frankia sp. G2 could fix nitrogen in vitro but could only infect Hippophae rhamnoides and not its host

plant, Casuarina equisetifolia. What the role of such ineffective strains is in nature is aquestion for the future.

Acknowledgements We thank Hubert A. Lechevalier for his constant

support of this project, Lee D. Simon, Gloria Bin- kowksi, and Ed Seling for their assistance with the SEM's, Thomas Dillon for help with serology and infectivity studies, and Magda Gagliardi for indispens- able assistance. This work was supported in part by the Charles and Johanna Busch Fund and grant Nos. 59-2341 -0-1-439-0 and 59-2502-0-1 -446-0 from the U.S. Department of Agriculture.

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Documents

Physiology, chemistry, serology, and infectivity of two Frankia isolates from Alnus incana subsp. rugosa