APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1991, p. 1540-1545 Vol. 57, No. 5 0099-2240/91/051540-06$02.00/0
Copyright ? 1991, American Society for Microbiology
Subgroups of the Cowpea Miscellany: Symbiotic Specificity within
Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus,
Arachis hypogaea, and Macroptilium atropurpureumt
JANICE E. THIES, B. BEN BOHLOOL,* AND PAUL W. SINGLETON
NifTAL Project, University of Hawaii, 1000 Holomua Avenue, Paia, Hawaii 96779-9744
Received 4 December 1990/Accepted 19 February 1991
Rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group. To delineate the
degree of specificity exhibited by four legumes in the cowpea miscellany, we tested the symbiotic characteristics of indigenous cowpea bradyrhizobia on cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea). The most-probable-number counts of indigenous bradyrhizobia at
three sites on Maui, Hawaii, were substantially different on the four hosts: highest on siratro, intermediate on cowpea, and significantly lower on both lima bean and peanut. Bradyrhizobia from single cowpea nodules from the most-probable-number assays were inoculated onto the four hosts. Effectiveness patterns of these rhizobia on cowpea followed a normal distribution but were strikingly different on the other legumes. The effectiveness profiles on siratro
and cowpea were similar but not identical. The indigenous cowpea-derived bradyrhizobia were of only moderate effectiveness on siratro and were in all cases lower than the inoculant-quality reference strain. Between 5 and 51 % of
the bradyrhizobia, depending on site, failed to nodulate peanut, whereas 0 to 32 % failed to nodulate lima bean. No significant correlation was observed between the relative effectiveness of the bradyrhizobia on cowpea and their corresponding effectiveness on either lima bean or peanut. At all sites, bradyrhizobia that were ineffective on cowpea but that effectively nodulated lima bean, peanut, or both were found. Eighteen percent or fewer of the bradyrhizobia were as effective on lima bean as the reference inoculant strain; 44% or fewer were as effective on peanut as the reference strain. Only 18% of all cowpea-derived bradyrhizobia tested were able to form N,-fixing nodules on both
lima bean and peanut. These results indicate the need to measure indigenous bradyrhizobial population characteristics
directly with the crop of interest to obtain an accurate assessment of the need to inoculate.
Rhizobial classification is based first and foremost on host specificity on the part of these hosts or whether overlap exists specificity. In the genus Bradyrhizobium, the bacteria that nodulate between the various groups. soybean are classified as Bradyrhizobium japonicum and all others Size and effectiveness of indigenous rhizobial populations are are assigned to the Bradyrhizobium spp., previously referred to as primary factors that determine the incidence and magnitude of cowpea rhizobia or more loosely as tropical rhizobia. This has been legume inoculation response (11, 14). Singleton and Tavares (11) the source of misconceptions about the promiscuity of tropical isolated indigenous bradyrhizobia from cowpea, lima bean, and legumes and the ubiquity of tropical bradyrhizobia (10) and has led peanut growing in four Hawaiian soil samples and tested the to false recommendations regarding the inoculation requirements of effectiveness of the bradyrhizobia on the legumes from which they tropical legumes. For example, cowpea (Vigna unguiculata), lima were isolated. They found that, within a soil sample, the range of bean (Phaseolus lunatus), peanut (Arachis hypogaea), and siratro effectiveness of indigenous rhizobial isolates for these hosts (Macroptilium atropurpureum) are all thought to nodulate with the differed. Their results indicated that considerable diversity in the cowpea rhizobia, now classified as Bradyrhizobium spp. But relative effectiveness of indigenous Bradyrhizobium spp. individual bradyrhizobial isolates may not be equally infective or populations on these legumes exists. Such population diversity is effective on these four host legumes. Observations of differential also reflected in measured differences in the sizes of indigenous effectiveness led Burton (2) to separate these hosts into bradyrhizobial populations capable of nodulating these legumes (11, effectiveness groupings within the cowpea miscellany. Cowpea and 14), differences in the ability of bradyrhizobia to compete with siratro were included in one group, while lima bean and peanut inoculant strains for nodulation of the different hosts (14), and composed separate groupings. Under this system, it is assumed that differences in the incidence and magnitude of inoculation responses legume hosts within a given effectiveness grouping have similar obtained on these hosts in the same soil samples (11, 14). bradyrhizobial requirements. While these divisions may be useful In their studies, Singleton and Tavares (11) did not characterize for making strain recommendations and formulating an inoculant, the effectiveness of indigenous bradyrhizobial isolates from any they fail to indicate the degree of one of the hosts on the others. Hence, the nature of observed differences in the range of effectiveness of these isolates could not
be determined. In this study, we examined the nature of these †differences by assessing the degrees of specificity, in terms of both * Corresponding author. Journal series no. 3521 of the Hawaii nodulation and effectiveness, exhibited by siratro, lima bean, and Institute of Tropical Agriculture and Human Resources. peanut
SYMBIOTIC SPECIFICITY OF BRADYRHIZOBIA FOR LEGUMES 1541 VOL. 57, 1991
TABLE 1. Location and characteristics of three sites on the island of Maui, Hawaii"
pH Legume genera Site no. Elevation Soil Median annual rainfall` (mm/year) and name (m) subgroup' present at site 6.8 Leucaena, Prosopis Torroxic 1, Hashimoto Farm 37 322 7.5 Leucaena, Indigofera, 375 2, Kula Agricultural Park 366 haplustoll Macroptilium, Torroxic Prosopis haplustoll 5.3 Desmodium, Trifolium, 3, Haleakala Station 660 Humoxic tropohumult 1,800 Acacia, Crotalaria ? For more detailed descriptions of these sites, see reference 18. b From reference 10. C From reference 3.
when inoculated with bradyrhizobia from cowpea nodules. Our each test host. Nodulation characteristics were recorded, and leaf
results further substantiate division of the cowpea miscellany into chlorophyll content (chlorophyll a plus chlorophyll b) on six leaf disks (diameter, 0.635 cm) per plant (7), taken from the most effectiveness subgroups, particularly as they relate to inoculation response, and illustrate that measurement of indigenous recently fully expanded trifoliate leaf 32 days after inoculation for Bradyrhizobium spp. population characteristics should be made cowpea and lima bean and 41 DAI for peanut and siratro, was determined. directly with the host of interest to obtain an accurate assessment of the symbiotic capability of the population. Data analysis. Effectiveness of individual nodule bradyrhizobia
in symbiosis with the test hosts was divided into four categories: highly effective, effective, moderately effective, and ineffective.
The bradyrhizobia were considered ineffective if the chlorophyll MATERIALS AND METHODS content of host plant leaf disks was within the 95% confidence interval for the chlorophyll content of uninoculated (nonnodulated) Soil sampling and enumeration of indigenous Bradyrhizo-control plants; moderately effective if host leaf disk chlorophyll bium spp. populations. Soil samples were collected from fallow content was higher than the upper confidence limit for areas at three field sites on the island of Maui, Hawaii (Table 1). uninoculated control plants but less than the lower confidence limit After the top 1 cm of soil was removed, 20 to 25 for chlorophyll content of plants nodulated by the reference strains 2.54-cm-diameter soil cores to a depth of 25 cm were taken in a TAL 658 for cowpea, peanut, and siratro and TAL 644 for lima grid pattern around each field area. Soil cores were pooled, mixed, bean; effective if leaf disk chlorophyll content was within the 95% subsampled for determination of moisture content, and stored at confidence interval; and highly effective if leaf chlorophyll content 4?C. The most-probable-number (MPN) of indigenous was higher than the upper confidence limit for chlorophyll content bradyrhizobia in each soil sample was determined (for four test of plants inoculated with the known reference strains listed above. hosts: V. unguiculata cv. Knuckle Purplehull, P. lunatus cv. Kendall tau b rank correlation analysis (8) of host leaf chlorophyll Henderson's Baby, A. hypogaea cv. Burpee Florunner, and M. content was used to assess the degree of relatedness between the atropurpureum cv. Siratro. Serial 1:2, 1:5, or 1:10 soil dilutions effectiveness of nodule occupants on cowpea and their corre-were prepared and inoculated onto four replicate test plants per sponding effectiveness on siratro, peanut, and lima bean. dilution growing in either plastic growth pouches (cowpea, lima
bean, and peanut) or test tubes (siratro) (13). Plants in pouches were kept supplied with an adequate volume of N-free nutrient
solution (9), except that micronutrients were supplied by adding a RESULTS commercial micronutrient mix (0.25 ml liter-1; Hawaiian Horticultural). Plants were scored for nodulation 21 to 28 days At all sites, MPN counts of indigenous Bradyrhizobium spp. after inoculation. The MPN counts were determined by using the were highest on siratro (Table 2). Counts of indigenous Most-Probable-Number Enumeration System (17). bradyrhizobia were 2.4- to 18.6-fold lower when cowpea was used Assay for the effectiveness of indigenous Bradyrhizobium spp. as the trap host. Population counts on peanut and lima bean were A representative sample of nodules was taken from each of the from 6- to more than 1,000-fold lower than those obtained on MPN assays performed on cowpea. Nodules were selected from siratro and from 2.3- to 62-fold lower than those obtained on all dilutions when present. Three or more replicate nodules per test cowpea. host formed by inoculant-quality reference strains TAL 644 (CIAT