Growth, persistence and competition between strains of Rhizobium lupini schroeter

Strains of Rhizobium lupini on Lupinus spp. and Ornithopus sativus (serradella) were tested for growth, persistence and competition in nodule formation in growth cabinets, under glass house conditions and in the field. The literature on methods of isolation, identification and enumeration was reviewed. The accuracy and convenience of various methods when handling large numbers of samples were assessed. The Miles and Misra drop plate method using a Colworth droplette apparatus with antibiotic resistant mutants isolated from effective and ineffective strains was the method chosen for enumeration of R. lupini in soil. The fluorescent antibody (FA) technique, with antisera specific for the strains used and a membrane filtration technique were superior to the drop plate method when numbers were low but were more time consuming. The plant infection technique was laborious and less sensitive than other methods. Numbers from membranes incubated on agar containing antibiotics and from the FA method were not significantly different. Strain identification in nodules by FA and the antibiotic resistant mutants agreed fully. Fixtures of strains in one nodule could be identified by these methods. Mutant strains were shown to remain stable after five plant passages. When a mixture of effective strains N.Z.P. 2243 and N.Z.P. 5214 was used as inoculum on L. angustifolius and O. sativus the former strain always formed more nodules. The ineffective strain P.D.D. 3152 formed more nodules than N.Z.P. 5052 but neither formed as many as the effective strains. Since R. lupini strains nodulated O. sativus and is a small plant it was used in growth cabinet studies. Nodules appeared sooner at 25° than at 17° or 30°. Effective strains nodulated earlier than ineffective ones. Added nitrogen up to 100 p.p.m. did not inhibit nodulation. O. sativus inoculated with strain N.Z.P. 5214 formed significantly more nodules at pH 5 to 7 than at 9 and 10. Strains were tested for their persistence in soil. The optimum temperature was 25° . Survival was better at field capacity than at 20% water holding capacity; more survived In gamma sterilized soil than field soil. In the field there was a more rapid decline than in the laboratory, in the summer death occurred sooner. As in laboratory experiments, mixtures of two strains declined faster. The number of nodules on test plants from strains added to the soil also declined with time. The proportion of nodules formed by each of two strains on test plants did not reflect the proportion in which the mixed inoculum had been applied to the soil. The ineffective strain was a poor competitor in the presence of other effective strains and always formed a small percentage of the nodules. When large numbers were added in the inoculum, a high proportion of nodules from added strains were formed. The host plant rhizosphere greatly stimulated the multiplication of rhizobia and even non-host plants, both legumes and non-legumes, caused some increase in numbers. Stereoscan examination of nodules showed marked differences between nodules formed by effective and ineffective strains.