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Small leguminosae as test plants for nodulation of Rhizobium leguminosarum and other rhizobia and agrobacteria harbouring a leguminosarum sym-plasmid
Plant Science Letters, 27 (1982) 317--325 EisevieF ScientificPublishers Ireland Ltd.
317
SMALL LEGUMINOSAE AS TEST PLANTS FOR NODULATION OF RHIZOBIUNI LEGUA4INOSAR UM AND OTHER RHIZOBIA AND AGROBACTERIA HARBOURING A LEGUMINOSARUM SYM-PLASMID
A.A.N. VAN BRUSSEL, T. TAK, A. WETSELAARa, E. PEES and C.A. WIJFFELMAN Botanical Laboratory, Research Group of Rhizobium Genetics and aHortus Botanicus, Nonnensteeg 3, 2311 VJ Leiden (The Netherlands) (Received January 12th, 1982) (Revision received April 13th, 1982) (Accepted May 12th, 1982)
SUMMARY Small plants from the pea cross-inoculation group were selected for rapid nodulation on agar in test tubes. Plants from the genus Vicia were best; strains of the species hirsuta, tetrasperma, sativa and lathyroides nodulated respectively 4, 5, 4 and 7 days after inoculation with Rhizobium leguminosarum RBL 1. Rapid nodulation, visibility of the root system and in situ acetylene reduction makes the culture tube system ideal for genetic studies o f Rh. leguminosarum. A number of bacteria including Agrobacterium tumefaciens containing a Sym.plssmid from Rh. leguminosarum formed root nodules. A Vicia sativa strain formed thick and short roots (Tsr) when inoculated with a bacterium harbouring a Sym-plasmid from Rh. legumino. sarum. Crown gall formation could also be investigated by wounding of axenic Vicia hirsuta in test tubes.
INTRODUCTION Rh. leguminosarum forms symbiotic, nitrogen fixing, nodules on roots of the so,ailed pea cross-inoculation group which comprises the genera Pisum, Vicia, Lathyrus and Lens [ 1]. Investigation of the genetic system of this root nodule symbiosis requires a simple test system for symbiotic properties giving rapid results. In our laboratory, as in others [2], tests on root-nodule formation have been performed with peas, but these are rather tall plants and thus unsuitable for screening large numbers of bacterial strains. MoreAbbreviations: Tsr, thick and short roots. 0304--4211/82/0000--0000/$02.75 © 1982 ElsevierScientific Publishers Ireland Ltd.
318
over peas do not nodulate quickly o n agar or liquid medium [2] and their roots must be protected from light. In contrast to peas, some Vicia species are very small when y o u n g and can be cultured in test tubes (180 × 18 ram) on agar [3]. In this study we report on the usefulness of a number of plant species o f the pea cross-inoculation group for screening large numbers of bacteria for nodulation characteristics o n agar in test tubes. METHODS
Bacterial strains (Table I) were maintained on medium A agar slants [4] (yeast extract, mannitol, glucose medium). Plants were cultured in test tubes (180 × 18 mm) on Jensen agar [5 ] (CaHPO4,1.0; K2HPO4, 0.2; MgSO4.7H20, 0.2; NaCI, 0.2; FeC13, 0.1; Difco Bacto-Agar, 10 g - 1-1; trace element soluTABLE I BACTERIAL STRAINS Strains obtained from *P.J.J. Hooykaas and **J.E. Beringer. Strain no.
Characteristics
Eh. leguminosarum RBL 1 RBL 101 RBL 1086 248** LPR 1902" LPR 1802" LPR 1802 pJBSJI a 1062"* T3** LPR 1105" PRE
wild t y p e (synonym A171) rough derivative o f RBL 1, N o d - (synonym LPR 180) rough derivative of RBL 1 wild t y p e RCR 1039, rif R RCR 1018, rif R rifR, kan R ura-14, trp-16, str-86 1062 pJBSJI; ura-14, trp-16, str-86, kan R RCR 1001, rifR wild t y p e
Rh. trifolii LPR LPR LPR LPR
5020* 5035* 5039* 5039 pJB5JI
RCR 5, str R LPR 5020 (pRtr5a::Tn5), str R LPR 5035, cured for p R t r 5 a : : T n 5 , str R str R, kan R
Rh. phaseoli 1233"* 1233 pJBSJI (a)** 1233 pJBSJI (h)**
wild t y p e pig*, kan R p i g ' , kan R
A. tumefaciens LBA LBA LBA LBA LBA
4301" 4301 pJBSJI 201" 201 pJBSJI 44 20 pJB5JI
AchS, T i ' , Rif R, RecTi-, Rif R , Rec-~ Kan R wild t y p e C58, Ti ÷ Ti*, kan R Ach5, Ti*, kan R
a pJB5JI is the Sym-plasmid of strain 248 ( p R L 1) with a Tn5 insertion in the gene for medium bacteriocin production.
319
tion, 2.5 ml • 1-1 ; pH 6.5. The trace element solution contained CuSO4.5H:O, 0.035; MnSO4.4H20, 0.61; ZnSO4.7H20, 0.097; H3BO3, 1.27; Na=MoO4.4H20, 0.398 h . 1-1 ). Seeds were surface sterilized by immersion (30 min) in concentrated sulphuric acid followed by rinsing with tap water and immersion in undiluted commercial bleach for 10--30 min. To recover the bleach at least 5 washes with sterile water during 30 rain were needed. The seeds were swollen by incubating for 4 h in sterile water and germinated on 1% Jensen agar plates. The latter were incubated upside down for at least 4 days at 4°C, followed by 2 days incubation at 20°C in the dark. For Y. hirsuta the incubation period at 4°C was n o t needed for good germination. Inoculation of the roots was done with 3--7~lay-old bacterial medium A agar cultures before transferring to Jensen agar slants. The tubes, placed in racks with four rows of 12 tubes, were incubated 1 day in darkness at 20°C and subsequently in growth chambers (20°C; 70% relative humidity). The light intensity at the table surface was approx. 20.000 lux (Philips TLF 60W/33 fluorescent tubes). The day-length was 16 h. Nitrogenase activity was measured by acetylene reduction [6]. Culture tubes containing plants on Jensen agar were capped with Suba seal rubber stoppers and 10% of the air was replaced by acetylene. Ethylene production was measured with a gas chromatograph. Reisolation of Rhizobia and Agrobacteria from root nodules was done according to Vincent [5]. Nodules were immersed in ethanol 96% (30 s), followed by incubation for 2--3 min in HCl~cidified HgC12 (1 g . 1-1 ). The nodules were washed 6 times with sterile water, crushed in B (minimal) medium [4] and finally plated on A [4] or TY medium (Difco trypton, 5; Difco yeast extract, 3; CaC12, 1 and Difco Bacto.Agar, 15 g . 1-1 ) on which Rhizobium as well Agrobacterium grow. Detached colonies (10--20) were then tested for their respective markers. RESULTS A number of plant species of the pea cross-inoculation group were investigated on their properties to nodulate on agar with Rh. I q u m i n o m r u m RBL 1. The number of days needed to form visible nodules after inoculation and the dimensions of the young plants in relation to their usefulness in test tubes appear in Table II. Pisum ~ t i v u m " R o n d o " , Lathyrus tuberosus and Lathyrus aphaca and some Vic~ species did not nodulate on agar, although the Pisum and Lathyrus strains nodulate on gravel [4]. The time of appearance of the first nodules on agar varied from species to species and was n o t always the same within the species, when seed samples of different origin were tested. Seeds of V. sativa and V. tetr~pcrma varied in size; the larger seeds gave rise to faster growing plants, which nodulated earlier. The most rapid nodulation was with V. h / r ~ t a (Table II and Fig. 2), which occ~ ~onally had nodules after 3 days; with 21 out of 30 seed samples, nodules were observed after 4 days and with the rest a few days later. With V. sati~ 2 out
320
TABLE II FIRST VISIBLE NODULES OF RHIZOBIUM LEGUMINOSARUM RBL 1 ON JENSEN AGAR --, no nodulation on agar; +, see text for explanation; *, depending on the seed sample. No seed samples tested
Plant
P. sativum L. "Little marvel" "Rondo" Lens culinaris Medicus nigricans (Bibl.) G o d r o n
Lathyrus tuberosus L. aphaca L. pratensis L.
First visible nodules (days after infection)
Size+ of young plants
1 1 1
20 12
Large Large Small
1 1 1 1
> 15 -12
Small Small Large Small
3 1
> 12 -
Small Small
33
4--6
13 2 1
5--20 5 7
Medium Small or m e d i u m * Large Small
15 1 2
4--18 7
Small orlarge* Large Small
> 12
Large
Vicia Sect. cracca S.G. Gray cracca L. tenuifolla R o t h Sect. ervum (L.) S.F. Gray hirsuta (L.) S.F. Gray
tetrasperma (L.) Schreber disperma DC. pubeseens (DC.) Sect. vicia sativa L. hybrida L. lathyroides L. Sect. faba (Miller) S.F. Gray narbonensis L.
1
o f 15 seed samples n o d u l a t e d in 4 days, 8 after 8--10 days and the remainder later. Some V. tetrasperma and the V. disperma plants p r o d u c e d their first nodules after 5 days. T he size o f th e plants is o f i m p o r t a n c e f o r culturing in test tubes. Small plants can be grown in test tubes f o r up t o 4 weeks w i t h o u t t he t u b e becoming t o o small f o r normal development. Medium size plants can also be grown f o r a similar period, b u t t he stem t ouc he s the c o t t o n plug after a b o u t a week and plant d e v e l o p m e n t is at least slightly inhibited. Plants are m e n t i o n e d large when their size makes culture in test tubes impossible. The sizes of plants w h e n nodules b e c o m e first visible are f r o m r o o t tip t o stem tip: for small 8 cm, f o r m e d i u m 10 cm and f or large ~ 1 4 cm. Because o f rapid nodulation, size and availability of large am ount s of h o m o g e n o u s seeds, V. hirsuta and V. sativa subsp, nigra (small size, Fig. 3) were chosen as test plants for genetic experiments, for nodul at i on and acetylene reduction. In Table III, the di f f er e nt n o d u l a t i o n capacity of bot h
321
,
600
P~
Mm.M
~ 2 ~
7
13
24
1~rne(days) Fig. 1. Acetylene reduction by single ~¢Jo hirsuta plants. Plants were inoculated at day O, with Rh. legumino~-um strairm.
Fig. 2. V. hirsuta plants cultured in test tubes. The plants were p h a t o ~ a t J h e d relc~ctivelv 2, 4, 7, t 4 and 21 days after inoculatio~ with Rh. ieguminosarum RIgid'l". ~ " Fig. 3. V. sativa plants cultured in test tubes. The plants were photographed in situ 2, 4, 7, 14 and 21 days after inoculation w i ~ Rh. leguminosarum PRE.
322
Fig. 4. V. hirsuta with an Ach 5 crown gall, produced by strain 4420 pJBSJI. The l~lant was wounded and infected 4 days after trander to Jemmn agar and photos~phed 2B days later. Fig. 5. V. sativa inoculated with Rh. leguminosarum 1802 (left) and 1802 pJBSJI (right). The pr~enee of pJBf~'I gives ri~ to Tit and ineffeetDe nodul~ in the axfles of some lateral roots. Plant8 were photographed 21 days after inoeulation.
plants is given. Most Rh. leguminosarum strains nodulate V. hirsuta as well as V. sativa, b u t the a m o u n t o f acetylene reduction differs between different bacterial strains; acetylene reduction b y individual plants is compared in Fig. 1. Some non-nodulating Rh. leguminosarum strains as ttBL 101 and L P R 1802 do n o t nodulate V/c/a as expected, b u t strain LPR 1 8 0 2 in contrast to R B L 101 became nodulating after the introduction of the Rh. leguminosarum Sym-plasmid pJBSJI on b o t h V. hirsuta and V. sativa. Some o f our mutants, as RBL 1086, do only nodutate on V: hirsuta. Rh. tr~oU~ and Rh. phaseoli do n o t nodulate b o t h Vicias, b u t the introduction of pJBSJI makes the strains nodulating on V. hirsuta and V. sw~iva. Of special interest are the A. tumefueisns strains LBA 4301 and LBA 201. A f t e r introduction o f pJBSJI by conjugation with strain T3, these strains formed ineffective nodules with V. sativa, b u t failed to nodulate V. hirs~ta or P. sativum
323
TABLE HI N O D U L A T I O N CAPACITY A N D A C E T Y L E N E R E D U C T I O N O F BACTERIA O N V. H I R S U T A A N D V. S A T I V A
V. himuta
Strain
V. sativa
Nodulation a
Acetylene
Nodula-
reduction
tion
b
a
Acetylene reductionb
Rh. legumincearum RBL 1 RBL 101 RBL 1086
+ . +
248
+ + . +
LPR 1902 LPR 1802 LPR 1802 pJBSJI 1062 T3 LPR 1105 PRE
+
+
+++
--
--
--
++ +
+ +
+++ + ND +++ +++ ND ND +
.
.
.
. -
.
.
+
-
+ +, +
ND +++ ++++
+ + + + +
+
ND ND -
-+
1 2 3 3
--
--
--
ND
1233 pJB5JI pig* 1233 pJBSJI pig-
+ +
+ +
+ +
ND ND
--
ND ND ND ND
-+ -+
-
Rh. trifolii LPR 5020 LPR 5039 LPR 5039 pJB5JI
Rh. phaseoli
A. tumefaciens LBA LBA LBA LBA
4301 4301 pJB5JI 201 201 pJBSJI
-
a Nodulation: +, nodules formed; - , no nodules formed. b Acetylene reduction: --, no significant reduction (< 10 nmol/plant/h); +, 10--50 nmol;
+ +, 5 0 - 1 0 0 nmol; + + +, 100---200 nmol, + + + +, more than 200 nmol plant -1 h -! ; ND, n o t determined. " R o n d o " . W h e n t h e y o u n g s t e m s o f V. hirsuta are w o u n d e d a n d i z d e c t e d w i t h A. tumefaciens, c o n t a i n i n g p J B S J I a n d a T i - p l a s m i d , c r o w n galls a r e f o r m e d (Fig. 4). W e u s e d t h i s p r o p e r t y a n d t h e o t h e r g e n e t i c m a r k e r s ( T a b l e I ) t o v e r i f y t h e g e n e t i c b a c k g r o u n d o f the A. tumefaciens sftvJns reisolated from the root nodules. A r e m a r k a b l e f e a t u r e o f V. setwa, w h i c h i t d o e s n o t s h a r e witk/~ V. hirsuta, is t h a t i t f o r m e d t h i c k e r a n d s h o r t e r r o o t s ( T s r ) i n t h e p r e s e n c e of Rhizo. bium and Agrobacterium t h a t c o n t a i n p J B S J I ( T a b l e m ) ; w i t h t h e s a m e strains without the plasmid much longer and thinner roots were formed ( F i g . 5).
324 DISCUSSION The usefulness of plants for nodulation tests with Rh. leguminosarum is mainly determined by two factors: (i) the time needed to form vmible nodules after inoculation and (ii) the ease of culture on agar in test tubes, which depends on the size of young plants. Some plants do not nodulate on agar; this is probably because of the light sensitivity of the roots. Some fast nodulating plants such as V. hirsuta and V. tetrasperma are very well suited for nodulation tests because of their size but the fast nodulating V. sativa and V. disperma species are too large. The slower nodulating V. sativa, V. lathyroides and V. pubescens, are also useful because of their small size. Lathyrus pratensis and Lens culinaris are slow to nodulate in test tubes but are of interest because they belong to other genera. Induction of nodules on one species of the pea cross-inoculation group by a bacterium does not necessarily signify that other plants of this group nodulate also with the same bacterium (Table II); thus one can easily overlook the presence and expression of the nodulation genes in a bacterium by using only one plant species to test nodulation. The capacity of nitrogen fixation (effectiveness) depends on both, bacterial strain and plant species used (Table III; Fig. 1). {This has been found earlier for several cross-inoculation ~roups [7]). The nature of the Tsr formed with strains that contain the plasmid pJB5JI has not been further investigated, but we use it as a marker for pJB5JI. Nodules formed by A. tumefaciens pJBSJI have been investigated by electron microscopy (in prep.). These are true nodules such as those produced on clover by A. tumefaciens with a Sym-plasmid of Rh. trifolii [8] and make V. sativa an interesting plant for nodulation tests. The choice of a plant species for screening large numbers of bacterial mutants for nodulation depends further on the availability of a large quantity of homogeneous seeds, ease of germination and of surface disinfection. V. hirsuta, disperma, tetrasperma and sat/va are self pollinated so that homogeneous seeds are easily obtained. We also investigated the petri dish culture system [9] for nodulation tests. This method is less laborious than the tube test system but plant development in petri dishes was inferior and nodulation usually occurred later. Virulence tests for Agrobacteriurn have previously been performed with much bigger plants [10] and not under anexic conditions; the V. hirsuta test system observed here combines the advantages of small plants with axenic conditions. ACKNOWLEDGEMENTS
The authors wish to acknowledge Mr. A. Engelman for sending a large amount of V. hirsuta seeds, Drs. G.J.C.M. van Vliet for advice and Drs. Clara Diaz for reading the English text.
325 REFERENCES 1 J.M. Vincent, Root nodule symbiosis with Rhizobium, in: A. Quispel (Ed.), The Biology of Nitrogen Fixation, North-Holland Publishing Co., Amsterdam, 1974, p. 265. 2 J.E. Beringer, J. Gen. Microbiol., 84 (1974) 188. 3 The Rothamsted Collection of Rhizobium, Catalogue of strains (1972). 4 A.A.N. van Bruuel, K. Planqu~ and A. Quispel, J. Gen. Microbiol., 101 (1977) 51. 5 J~M. Vincent, IBP Handbook no. 15, Blackwell Scientific Publications, Oxford and Edinburgh, 1970, p. 75 and p. 7. 6 R.W.F. Hardy, R.D. Holsten, E.K. Jackson and R.C. Burns, Plant Physiol., 43 (1968) 1185. 7 J.M. Vincent, Proc. Linnean Soc. New South Wales, 87 (1962) 8. 8 P J J , Hooykaas, A.A.N. van Bruuel, H. den Dulk-Ras, G.M.S. van Siogteren and R.A. Schilperoort, Nature, 291 (1981) 351. 9 B.G. Rolfe, P.M. Greuhoff and J. Shine, Plant Sci. Lett., 19 (1980) 277. 10 G.H. Bomhoff, P.M. Klapwijk, H.C.M. Kester, R.A. Schilperoort, J.P. Hernaisteens and O. ScheU, Mol. Gen. Genet., 145 (1976) 177.
317
SMALL LEGUMINOSAE AS TEST PLANTS FOR NODULATION OF RHIZOBIUNI LEGUA4INOSAR UM AND OTHER RHIZOBIA AND AGROBACTERIA HARBOURING A LEGUMINOSARUM SYM-PLASMID
A.A.N. VAN BRUSSEL, T. TAK, A. WETSELAARa, E. PEES and C.A. WIJFFELMAN Botanical Laboratory, Research Group of Rhizobium Genetics and aHortus Botanicus, Nonnensteeg 3, 2311 VJ Leiden (The Netherlands) (Received January 12th, 1982) (Revision received April 13th, 1982) (Accepted May 12th, 1982)
SUMMARY Small plants from the pea cross-inoculation group were selected for rapid nodulation on agar in test tubes. Plants from the genus Vicia were best; strains of the species hirsuta, tetrasperma, sativa and lathyroides nodulated respectively 4, 5, 4 and 7 days after inoculation with Rhizobium leguminosarum RBL 1. Rapid nodulation, visibility of the root system and in situ acetylene reduction makes the culture tube system ideal for genetic studies o f Rh. leguminosarum. A number of bacteria including Agrobacterium tumefaciens containing a Sym.plssmid from Rh. leguminosarum formed root nodules. A Vicia sativa strain formed thick and short roots (Tsr) when inoculated with a bacterium harbouring a Sym-plasmid from Rh. legumino. sarum. Crown gall formation could also be investigated by wounding of axenic Vicia hirsuta in test tubes.
INTRODUCTION Rh. leguminosarum forms symbiotic, nitrogen fixing, nodules on roots of the so,ailed pea cross-inoculation group which comprises the genera Pisum, Vicia, Lathyrus and Lens [ 1]. Investigation of the genetic system of this root nodule symbiosis requires a simple test system for symbiotic properties giving rapid results. In our laboratory, as in others [2], tests on root-nodule formation have been performed with peas, but these are rather tall plants and thus unsuitable for screening large numbers of bacterial strains. MoreAbbreviations: Tsr, thick and short roots. 0304--4211/82/0000--0000/$02.75 © 1982 ElsevierScientific Publishers Ireland Ltd.
318
over peas do not nodulate quickly o n agar or liquid medium [2] and their roots must be protected from light. In contrast to peas, some Vicia species are very small when y o u n g and can be cultured in test tubes (180 × 18 ram) on agar [3]. In this study we report on the usefulness of a number of plant species o f the pea cross-inoculation group for screening large numbers of bacteria for nodulation characteristics o n agar in test tubes. METHODS
Bacterial strains (Table I) were maintained on medium A agar slants [4] (yeast extract, mannitol, glucose medium). Plants were cultured in test tubes (180 × 18 mm) on Jensen agar [5 ] (CaHPO4,1.0; K2HPO4, 0.2; MgSO4.7H20, 0.2; NaCI, 0.2; FeC13, 0.1; Difco Bacto-Agar, 10 g - 1-1; trace element soluTABLE I BACTERIAL STRAINS Strains obtained from *P.J.J. Hooykaas and **J.E. Beringer. Strain no.
Characteristics
Eh. leguminosarum RBL 1 RBL 101 RBL 1086 248** LPR 1902" LPR 1802" LPR 1802 pJBSJI a 1062"* T3** LPR 1105" PRE
wild t y p e (synonym A171) rough derivative o f RBL 1, N o d - (synonym LPR 180) rough derivative of RBL 1 wild t y p e RCR 1039, rif R RCR 1018, rif R rifR, kan R ura-14, trp-16, str-86 1062 pJBSJI; ura-14, trp-16, str-86, kan R RCR 1001, rifR wild t y p e
Rh. trifolii LPR LPR LPR LPR
5020* 5035* 5039* 5039 pJB5JI
RCR 5, str R LPR 5020 (pRtr5a::Tn5), str R LPR 5035, cured for p R t r 5 a : : T n 5 , str R str R, kan R
Rh. phaseoli 1233"* 1233 pJBSJI (a)** 1233 pJBSJI (h)**
wild t y p e pig*, kan R p i g ' , kan R
A. tumefaciens LBA LBA LBA LBA LBA
4301" 4301 pJBSJI 201" 201 pJBSJI 44 20 pJB5JI
AchS, T i ' , Rif R, RecTi-, Rif R , Rec-~ Kan R wild t y p e C58, Ti ÷ Ti*, kan R Ach5, Ti*, kan R
a pJB5JI is the Sym-plasmid of strain 248 ( p R L 1) with a Tn5 insertion in the gene for medium bacteriocin production.
319
tion, 2.5 ml • 1-1 ; pH 6.5. The trace element solution contained CuSO4.5H:O, 0.035; MnSO4.4H20, 0.61; ZnSO4.7H20, 0.097; H3BO3, 1.27; Na=MoO4.4H20, 0.398 h . 1-1 ). Seeds were surface sterilized by immersion (30 min) in concentrated sulphuric acid followed by rinsing with tap water and immersion in undiluted commercial bleach for 10--30 min. To recover the bleach at least 5 washes with sterile water during 30 rain were needed. The seeds were swollen by incubating for 4 h in sterile water and germinated on 1% Jensen agar plates. The latter were incubated upside down for at least 4 days at 4°C, followed by 2 days incubation at 20°C in the dark. For Y. hirsuta the incubation period at 4°C was n o t needed for good germination. Inoculation of the roots was done with 3--7~lay-old bacterial medium A agar cultures before transferring to Jensen agar slants. The tubes, placed in racks with four rows of 12 tubes, were incubated 1 day in darkness at 20°C and subsequently in growth chambers (20°C; 70% relative humidity). The light intensity at the table surface was approx. 20.000 lux (Philips TLF 60W/33 fluorescent tubes). The day-length was 16 h. Nitrogenase activity was measured by acetylene reduction [6]. Culture tubes containing plants on Jensen agar were capped with Suba seal rubber stoppers and 10% of the air was replaced by acetylene. Ethylene production was measured with a gas chromatograph. Reisolation of Rhizobia and Agrobacteria from root nodules was done according to Vincent [5]. Nodules were immersed in ethanol 96% (30 s), followed by incubation for 2--3 min in HCl~cidified HgC12 (1 g . 1-1 ). The nodules were washed 6 times with sterile water, crushed in B (minimal) medium [4] and finally plated on A [4] or TY medium (Difco trypton, 5; Difco yeast extract, 3; CaC12, 1 and Difco Bacto.Agar, 15 g . 1-1 ) on which Rhizobium as well Agrobacterium grow. Detached colonies (10--20) were then tested for their respective markers. RESULTS A number of plant species of the pea cross-inoculation group were investigated on their properties to nodulate on agar with Rh. I q u m i n o m r u m RBL 1. The number of days needed to form visible nodules after inoculation and the dimensions of the young plants in relation to their usefulness in test tubes appear in Table II. Pisum ~ t i v u m " R o n d o " , Lathyrus tuberosus and Lathyrus aphaca and some Vic~ species did not nodulate on agar, although the Pisum and Lathyrus strains nodulate on gravel [4]. The time of appearance of the first nodules on agar varied from species to species and was n o t always the same within the species, when seed samples of different origin were tested. Seeds of V. sativa and V. tetr~pcrma varied in size; the larger seeds gave rise to faster growing plants, which nodulated earlier. The most rapid nodulation was with V. h / r ~ t a (Table II and Fig. 2), which occ~ ~onally had nodules after 3 days; with 21 out of 30 seed samples, nodules were observed after 4 days and with the rest a few days later. With V. sati~ 2 out
320
TABLE II FIRST VISIBLE NODULES OF RHIZOBIUM LEGUMINOSARUM RBL 1 ON JENSEN AGAR --, no nodulation on agar; +, see text for explanation; *, depending on the seed sample. No seed samples tested
Plant
P. sativum L. "Little marvel" "Rondo" Lens culinaris Medicus nigricans (Bibl.) G o d r o n
Lathyrus tuberosus L. aphaca L. pratensis L.
First visible nodules (days after infection)
Size+ of young plants
1 1 1
20 12
Large Large Small
1 1 1 1
> 15 -12
Small Small Large Small
3 1
> 12 -
Small Small
33
4--6
13 2 1
5--20 5 7
Medium Small or m e d i u m * Large Small
15 1 2
4--18 7
Small orlarge* Large Small
> 12
Large
Vicia Sect. cracca S.G. Gray cracca L. tenuifolla R o t h Sect. ervum (L.) S.F. Gray hirsuta (L.) S.F. Gray
tetrasperma (L.) Schreber disperma DC. pubeseens (DC.) Sect. vicia sativa L. hybrida L. lathyroides L. Sect. faba (Miller) S.F. Gray narbonensis L.
1
o f 15 seed samples n o d u l a t e d in 4 days, 8 after 8--10 days and the remainder later. Some V. tetrasperma and the V. disperma plants p r o d u c e d their first nodules after 5 days. T he size o f th e plants is o f i m p o r t a n c e f o r culturing in test tubes. Small plants can be grown in test tubes f o r up t o 4 weeks w i t h o u t t he t u b e becoming t o o small f o r normal development. Medium size plants can also be grown f o r a similar period, b u t t he stem t ouc he s the c o t t o n plug after a b o u t a week and plant d e v e l o p m e n t is at least slightly inhibited. Plants are m e n t i o n e d large when their size makes culture in test tubes impossible. The sizes of plants w h e n nodules b e c o m e first visible are f r o m r o o t tip t o stem tip: for small 8 cm, f o r m e d i u m 10 cm and f or large ~ 1 4 cm. Because o f rapid nodulation, size and availability of large am ount s of h o m o g e n o u s seeds, V. hirsuta and V. sativa subsp, nigra (small size, Fig. 3) were chosen as test plants for genetic experiments, for nodul at i on and acetylene reduction. In Table III, the di f f er e nt n o d u l a t i o n capacity of bot h
321
,
600
P~
Mm.M
~ 2 ~
7
13
24
1~rne(days) Fig. 1. Acetylene reduction by single ~¢Jo hirsuta plants. Plants were inoculated at day O, with Rh. legumino~-um strairm.
Fig. 2. V. hirsuta plants cultured in test tubes. The plants were p h a t o ~ a t J h e d relc~ctivelv 2, 4, 7, t 4 and 21 days after inoculatio~ with Rh. ieguminosarum RIgid'l". ~ " Fig. 3. V. sativa plants cultured in test tubes. The plants were photographed in situ 2, 4, 7, 14 and 21 days after inoculation w i ~ Rh. leguminosarum PRE.
322
Fig. 4. V. hirsuta with an Ach 5 crown gall, produced by strain 4420 pJBSJI. The l~lant was wounded and infected 4 days after trander to Jemmn agar and photos~phed 2B days later. Fig. 5. V. sativa inoculated with Rh. leguminosarum 1802 (left) and 1802 pJBSJI (right). The pr~enee of pJBf~'I gives ri~ to Tit and ineffeetDe nodul~ in the axfles of some lateral roots. Plant8 were photographed 21 days after inoeulation.
plants is given. Most Rh. leguminosarum strains nodulate V. hirsuta as well as V. sativa, b u t the a m o u n t o f acetylene reduction differs between different bacterial strains; acetylene reduction b y individual plants is compared in Fig. 1. Some non-nodulating Rh. leguminosarum strains as ttBL 101 and L P R 1802 do n o t nodulate V/c/a as expected, b u t strain LPR 1 8 0 2 in contrast to R B L 101 became nodulating after the introduction of the Rh. leguminosarum Sym-plasmid pJBSJI on b o t h V. hirsuta and V. sativa. Some o f our mutants, as RBL 1086, do only nodutate on V: hirsuta. Rh. tr~oU~ and Rh. phaseoli do n o t nodulate b o t h Vicias, b u t the introduction of pJBSJI makes the strains nodulating on V. hirsuta and V. sw~iva. Of special interest are the A. tumefueisns strains LBA 4301 and LBA 201. A f t e r introduction o f pJBSJI by conjugation with strain T3, these strains formed ineffective nodules with V. sativa, b u t failed to nodulate V. hirs~ta or P. sativum
323
TABLE HI N O D U L A T I O N CAPACITY A N D A C E T Y L E N E R E D U C T I O N O F BACTERIA O N V. H I R S U T A A N D V. S A T I V A
V. himuta
Strain
V. sativa
Nodulation a
Acetylene
Nodula-
reduction
tion
b
a
Acetylene reductionb
Rh. legumincearum RBL 1 RBL 101 RBL 1086
+ . +
248
+ + . +
LPR 1902 LPR 1802 LPR 1802 pJBSJI 1062 T3 LPR 1105 PRE
+
+
+++
--
--
--
++ +
+ +
+++ + ND +++ +++ ND ND +
.
.
.
. -
.
.
+
-
+ +, +
ND +++ ++++
+ + + + +
+
ND ND -
-+
1 2 3 3
--
--
--
ND
1233 pJB5JI pig* 1233 pJBSJI pig-
+ +
+ +
+ +
ND ND
--
ND ND ND ND
-+ -+
-
Rh. trifolii LPR 5020 LPR 5039 LPR 5039 pJB5JI
Rh. phaseoli
A. tumefaciens LBA LBA LBA LBA
4301 4301 pJB5JI 201 201 pJBSJI
-
a Nodulation: +, nodules formed; - , no nodules formed. b Acetylene reduction: --, no significant reduction (< 10 nmol/plant/h); +, 10--50 nmol;
+ +, 5 0 - 1 0 0 nmol; + + +, 100---200 nmol, + + + +, more than 200 nmol plant -1 h -! ; ND, n o t determined. " R o n d o " . W h e n t h e y o u n g s t e m s o f V. hirsuta are w o u n d e d a n d i z d e c t e d w i t h A. tumefaciens, c o n t a i n i n g p J B S J I a n d a T i - p l a s m i d , c r o w n galls a r e f o r m e d (Fig. 4). W e u s e d t h i s p r o p e r t y a n d t h e o t h e r g e n e t i c m a r k e r s ( T a b l e I ) t o v e r i f y t h e g e n e t i c b a c k g r o u n d o f the A. tumefaciens sftvJns reisolated from the root nodules. A r e m a r k a b l e f e a t u r e o f V. setwa, w h i c h i t d o e s n o t s h a r e witk/~ V. hirsuta, is t h a t i t f o r m e d t h i c k e r a n d s h o r t e r r o o t s ( T s r ) i n t h e p r e s e n c e of Rhizo. bium and Agrobacterium t h a t c o n t a i n p J B S J I ( T a b l e m ) ; w i t h t h e s a m e strains without the plasmid much longer and thinner roots were formed ( F i g . 5).
324 DISCUSSION The usefulness of plants for nodulation tests with Rh. leguminosarum is mainly determined by two factors: (i) the time needed to form vmible nodules after inoculation and (ii) the ease of culture on agar in test tubes, which depends on the size of young plants. Some plants do not nodulate on agar; this is probably because of the light sensitivity of the roots. Some fast nodulating plants such as V. hirsuta and V. tetrasperma are very well suited for nodulation tests because of their size but the fast nodulating V. sativa and V. disperma species are too large. The slower nodulating V. sativa, V. lathyroides and V. pubescens, are also useful because of their small size. Lathyrus pratensis and Lens culinaris are slow to nodulate in test tubes but are of interest because they belong to other genera. Induction of nodules on one species of the pea cross-inoculation group by a bacterium does not necessarily signify that other plants of this group nodulate also with the same bacterium (Table II); thus one can easily overlook the presence and expression of the nodulation genes in a bacterium by using only one plant species to test nodulation. The capacity of nitrogen fixation (effectiveness) depends on both, bacterial strain and plant species used (Table III; Fig. 1). {This has been found earlier for several cross-inoculation ~roups [7]). The nature of the Tsr formed with strains that contain the plasmid pJB5JI has not been further investigated, but we use it as a marker for pJB5JI. Nodules formed by A. tumefaciens pJBSJI have been investigated by electron microscopy (in prep.). These are true nodules such as those produced on clover by A. tumefaciens with a Sym-plasmid of Rh. trifolii [8] and make V. sativa an interesting plant for nodulation tests. The choice of a plant species for screening large numbers of bacterial mutants for nodulation depends further on the availability of a large quantity of homogeneous seeds, ease of germination and of surface disinfection. V. hirsuta, disperma, tetrasperma and sat/va are self pollinated so that homogeneous seeds are easily obtained. We also investigated the petri dish culture system [9] for nodulation tests. This method is less laborious than the tube test system but plant development in petri dishes was inferior and nodulation usually occurred later. Virulence tests for Agrobacteriurn have previously been performed with much bigger plants [10] and not under anexic conditions; the V. hirsuta test system observed here combines the advantages of small plants with axenic conditions. ACKNOWLEDGEMENTS
The authors wish to acknowledge Mr. A. Engelman for sending a large amount of V. hirsuta seeds, Drs. G.J.C.M. van Vliet for advice and Drs. Clara Diaz for reading the English text.
325 REFERENCES 1 J.M. Vincent, Root nodule symbiosis with Rhizobium, in: A. Quispel (Ed.), The Biology of Nitrogen Fixation, North-Holland Publishing Co., Amsterdam, 1974, p. 265. 2 J.E. Beringer, J. Gen. Microbiol., 84 (1974) 188. 3 The Rothamsted Collection of Rhizobium, Catalogue of strains (1972). 4 A.A.N. van Bruuel, K. Planqu~ and A. Quispel, J. Gen. Microbiol., 101 (1977) 51. 5 J~M. Vincent, IBP Handbook no. 15, Blackwell Scientific Publications, Oxford and Edinburgh, 1970, p. 75 and p. 7. 6 R.W.F. Hardy, R.D. Holsten, E.K. Jackson and R.C. Burns, Plant Physiol., 43 (1968) 1185. 7 J.M. Vincent, Proc. Linnean Soc. New South Wales, 87 (1962) 8. 8 P J J , Hooykaas, A.A.N. van Bruuel, H. den Dulk-Ras, G.M.S. van Siogteren and R.A. Schilperoort, Nature, 291 (1981) 351. 9 B.G. Rolfe, P.M. Greuhoff and J. Shine, Plant Sci. Lett., 19 (1980) 277. 10 G.H. Bomhoff, P.M. Klapwijk, H.C.M. Kester, R.A. Schilperoort, J.P. Hernaisteens and O. ScheU, Mol. Gen. Genet., 145 (1976) 177.