Canadian Journal of Microbiology. 13(7): 910-913 (1967)
RHIZOBIAL RELATIONSHIP OF THE GENUS PHASEOLUS. I. CROSS-INOCULATION PERFORMANCE
OF DIFFERENT SPECIES TO R. PHASEOLI, R. JAPONICUM, AND THE COWPEA ORGANISM1

M. BARUA2 AND P. N. BHADURI3

1 Contribution supported financially by the Council of Scientific and Industrial Research, India.
2 Department of Botany, Presidency College, Calcutta-12, India.
3 Department of Botany, University of Burdwan, West Bengal, India.
4 This strain was kindly supplied by Dr. J. Brockwell through the courtesy of Dr. E. A. Schwinghamer, C.S.I.R.O., Australia.

Our knowledge of the cross-inoculation relationship within different species of the genus Phaseolus is still imperfect (Nutman 1956). Available reports indicate that the different species may be treated under two broad categories: first, those which are nodulated by Rhizobium phaseoli, and second, those like lima bean, which do not form nodules with it (Richmond 1926; Hofmann 1927; Fred, Baldwin, and McCoy 1932; Harris 1953). A distinction between the organisms of lima bean and Phaseolus vulgaris, respectively, of the "slow-growing" and "fast-growing" types has often been made (Jensen 1958).

Phaseolus aureus (green gram) and Phaseolus mungo (black gram) are extensively cultivated as major pulses in India and these two species formed abundant nodules in our experimental field where P. vulgaris failed to nodulate. This preliminary observation suggested that the microorganisms responsible for nodulation in green gram and French bean might be different. It is, therefore, of considerable interest to know the bacterial relationship of the various species grown in India.

Ten species of Phaseolus were tested as hosts. The bacterial cultures used were single colony isolations from a field collection of effective nodules. For R. phaseoli, strain CC5114, effective on P. vulgaris, was used throughout. The plants were grown in half-pint milk bottles containing washed and dried sand. The nitrogen-free mineral salts solution and the micronutrients as well as the method of culture were those employed by Schwinghamer (1960) for large-seeded plants. Surface-sterilized seeds were allowed to imbibe sterile water for 3-4 hours and then transferred aseptically to sterilized sand medium. Two plants were raised per bottle and they were maintained under greenhouse conditions. Bacterial suspension was added as soon as the seeds germinated and also 1 week later. Control cultures, without inoculum, were included in each set of experiments. The nutrient solution was replenished, as needed, avoiding cross-contamination. Nodulation was examined after 6 weeks' growth of the plants and each experiment was repeated for confirmation of the results.

Four of the species of Rhizobium, namely, R. leguminosarum, R. meliloti, R. trifolii, and R. lupini, isolated respectively from effective nodules of Pisum sativum, Melilotus indica, Trifolium alexandrinum, and Lupinus angustifolius proved non-nodulating when tested against the various species of Phaseolus. P. vulgaris, formed nodules only with R. phaseoli, a microorganism which was totally non-infective for soybean and cowpea and for the remaining nine species of Phaseolus. All the latter species of Phaseolus, on the other hand,

formed a homogeneous group. Between the members of this group cross-inoculation and reciprocal inoculation were successful and, therefore, no distinction could be made between these nodule microorganisms. This suggests a division of the species of Phaseolus into two groups on the basis of their rhizobial affinities, the "vulgaris" group nodulating with R. phaseoli and the "aureus" group forming nodules with Rhizobium sp. Cross-inoculation between the members of these two groups was, in every case, unsuccessful. Kvasnikov and Dolgikh (1955) noted a similar barrier of resistance between (a) soybean and P. vulgaris and (b) P. aureus and P. vulgaris which was not broken through grafting. P. aureus and P. vulgaris are, therefore, sufficiently distinct from each other in their rhizobial affinities. In the present study, R. phaseoli behaved as a strain-specific organism, being restricted in its infectivity only to P. vulgaris, whereas the P. aureus microorganism involved a large number of species of Phaseolus, both cultivated and wild, and could also nodulate soybean and cowpea.

Rhizobium japonicum from soybean nodules, when tested against the Phaseolus species of the "aureus" group, proved to be as infective as the strains of rhizobia isolated directly from the Phaseolus species themselves. Nearly identical results were obtained for the reverse inoculations on soybean hosts. The soybean microorganism, like those of the P. aureus group, was noninfective on P. vulgaris and the reverse inoculation also failed. Somewhat irregular results were obtained with cowpea inoculum. It failed to nodulate both P. vulgaris and Glycine max, but was very infective towards its own host. Against P. aureus and other species of Phaseolus of the same group, the nodulation performance was rather poor. Reciprocal inoculation of cowpea host was successful with rhizobia from soybean and P. aureus, but not with R. phaseoli. The results of cross-inoculation are summarized in Table I.

A close relationship between the P. aureus microorganism, on one hand, and R. japonicum from soybean and Rhizobium sp. from cowpea, on the other, is indicated in the present study. This observation is particularly interesting because of the observation of Leonard (1923) that a somewhat one-sided relationship exists between the soybean and cowpea nodule bacteria. In every instance, the soybean organism isolated from a large number of varieties produced nodules on cowpea plants, but bacteria obtained from different cowpea varieties gave inconsistent results on soybean hosts, characterized by numerous cases of failure to produce nodules. A similar non-reciprocal cross-inoculation has also been observed in the present investigation. The erratic performance of cowpea inocula to soybean hosts is not fully understood at present, but it appears that the response is conditioned by the nature of inoculum obtained from cowpea nodules. It is evident from the present investigation and the study of Leonard (1923) that cowpea plants could be nodulated by R. japonicum (nodulating soybean) and Rhizobium sp. (nodulating P. aureus), besides its own specific organism, and this is also probably true in nature. If isolation is made from a cowpea nodule inhabited by the soybean strain of R. japonicum, it may nodulate both cowpea and soybean. On the contrary, if the real cowpea strain is non-infective against soybean, nodule failure will result in soybean plant with inoculum obtained from cowpea nodules inhabited by the cowpea strain of Rhizobium sp.

TABLE I
Cross-inoculation relationship within Phaseolus vulgaris, Glycine max, Vigna sinensis, and different species of the Phaseolus aureus group

  Source of Rhizobium
Host Soybean Cowpea P. aureus P. mungo P. calcaratus P. sublobatus P. trilobus P. lathyroides P. ricciardianus P. atropurpureus P. aconitifolius
Glycine max (L.) Merr. 94-7* 0-0 100-7 94-5 100-5 96-7 100-6 88-4 96-5 85-6 85-4
Vigna sinensis Endl. 77-5 96-11 95-9 95-7 88-5 92-4 94-5 88-3 100-6 89-5 80-5
Phaseolus aureus Roxb. (var. NP 28) 94-7 83-5 96-10 80-8 100-6 94-5 100-9 87-4 93-6 93-6 85-5
Phaseolus mungo Linn. 91-6 75-3 100-7 100-6 85-2 91-5 100-8 93-5 96-6 100-6 92-5
Phaseolus calcaratus Roxb. 96-8 82-4 100-8 93-3 96-8 100-9 98-6 91-4 100-6 93-5 92-5
Phaseolus sublobatus Roxb. 100 5 90-3 100-5 85-5 100-9 97-8 93-6 100-5 89-5 89-6 94-7
Phaseolus trilobus Ait. 98-6 64-2 96-6 85-7 93-5 98-7 100-7 87-3 96-7 100-5 93-5
Phaseolus lathyroides Linn. 90-3 78-3 87-3 83-5 92-4 89-4 78-3 92-6 100-5 78-3 93-4
Phaseolus ricciardianus Ten. 98-8 86-3 100-6 90-5 96-6 100-5 100-6 98-S 96-6 100-6 95-5
Phaseolus atropurpureus (Moc. & Sesse) ex DC. Prod. 94 5 88-3 90-5 97-5 80-4 84-5 91-5 82-3 96-6 100-6 86-3
Phaseolus aconitifolius Jacq. 84-3 78-2 94-4 100-4 92-3 89-5 57-4 78-2 100-5 89-2 95-5
*With rare exceptions, 40-60 replicates of each host were exposed to each Rhizobium strain; the percentage of nodulated plants and the average lumber of nodules per plant are respectively denoted by the first and second figures (eliminating decimals) in each column. The corresponding figures are 91-9 for Phaseolus vulgaris Linn. inoculated with R. phaseoli strain CC511. No cross-reaction with any other groups occurred with the pair P. vulgaris and R. phaseoli.

NOTES

The position of the P. aureus group of rhizobia is, therefore, such that it is very closely related to both the soybean and cowpea bacteria, the relationship in each case being closer than that existing between the latter two organisms. The species of Phaseolus represented by P. aureus may function as bridging hosts (Vincent 1962) between soybean and cowpea, within which non-reciprocal cross-inoculation is known to exist. Considering that Phaseolus is a large genus having nearly 200 species, more information is necessary before the relative preponderance of species nodulating respectively with R. phaseoli and Rhizobium sp. (nodulating the P. aureus complex) can be estimated. Existing knowledge, and data from the present study, however, indicate that by far the majority of the investigated species of Phaseolus nodulate with bacteria akin to R. japonicum inhabiting soybean nodules, and that R. phaseoli, though usually regarded as a separate bacterial species, plays a very minor role in nodulation of the genus Phaseolus.

Acknowledgment
M. B. expresses her gratitude to Prof. H. C. Ganguleo, D.Sc., for the use of laboratory facilities at Presidency College.

RECEIVED AUGUST 30, 1966.
DEPARTMENT OF BOTANY, PRESIDENCY COLLEGE,
CALCUTTA, INDIA.