A Molecular approach to monitor persistence and spread of arbuscular mycorrizal inoculants in calliandra colathyrsus (meisn.)

Abstract

Calliandra calothyrsus is an important agroforestry tree species which requires inoculation with efficient arbuscular mycorrhiza fungal (AMF) inoculants. Morphological similarities of these inoculants hinder their precise identification. Identification and monitoring of persistence and spread of these inoculants subsequent to field inoculation require molecular approaches. It was hypothesized that probes can be designed, genetic diversity determined and infection levels evaluated for these inoculants. The objectives of this study were to develop specific DNA probes for one of the inoculants, determine genetic diversity of AMF and evaluate growth and infection levels of Calliandra calothyrsus. DNA probes were developed by utilizing polymerase chain reaction (PCR) and DNA sequencing techniques. The D2 region of the large sub unit of ribosomal DNA was amplified using P CR. The a mplicons were cloned 0 n vectors, sequenced, analysed for diversity and probes manually designed. A 12-week Calliandra calothyrsus glasshouse inoculation experiment was concurrently set using three AMF inoculants and their permuted mixtures; Scutellospora calospora (BEG 174) Gigaspora albida (BEG 172) and Glomus etunicatum (BEG 176). The AMF inoculants were compared for colonisation, phosphatase enzyme activity and their effects on growth of Calliandra calothyrsus. Three PCR primers were designed as probes and two of them were able to discriminate Scutellospora Calospora (BEG 174) from another Scutellospora Calospora isolate from a pot culture. In contrast, none of the two probes amplified DNA from the other AMF inoculants such as Gigaspora albida (BEG 172) and Glomus etunicatum (BEG 176). Results on molecular variance of the D2 region revealed very low genetic diversity among the AMF. The three AMF species resulted in varied levels of infection but had similar effects on the growth of Calliandra. Colonisation and growth response from mixed and single inoculants were also similar. Infection levels of between 11% to 54% (Trypan blue) and 9% to 74% (Alkaline phosphatase) were achieved and Calliandra response to inoculation varied from nodulation enhancement to stem biomass suppression. This study has demonstrated that the D2 domain of rDNA contains some variability adequate for the design of AMF isolate specific PCR probes but the variability is very low for studies of diversity across species. The study has also shown that some single AMF inoculants can result in higher root colonisation than some mixed inoculants. I f these probes are utilized, farmers c an benefit by knowing low long this inoculant will last in the soil, how often to inoculate and probably how far it can spread. The alkaline phosphatase enzyme assay method will also enhance studies of phosphorus uptake to reveal in situ phosphatase activity.