Close Association of Azospirillum and Diazotrophic Rods with Different Root Zones of Kallar Grass

Abstract

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloafusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N2-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by 15N2 incorporation. On the rhizoplane we found 2.0 x 107 diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCl treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 x 107 cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 x 104 nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1 %, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.

Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races

“…In contrast to opportunistic endophytes (Reinhold-Hurek and Hurek, 1998b), several diazotrophic endophytes, such as the plant-associated Azoarcus species, Acetobacter diazotrophicus or Herbaspirillum seropedicae, appear to be tightly associated with plant-associated fungi (Hurek et al, 1997b) or plants: they have not been isolated from soil or cannot survive well in it (Reinhold et al, 1986;Reinhold-Hurek et al, 1993a;Boddey, 1995;James and Olivares, 1998). In contrast, two newly described species of Azoarcus, A. tolulyticus (Zhou et al, 1995) and A. evansii (Anders et al, 1995), have been found exclusively in soil.…”

“…Plant roots were thoroughly washed with sterile water and surface sterilized with sodium hypochloride (Reinhold et al, 1986). Weighed roots were either homogenized by grinding with mortar and pestle in a standardized volume of buffer g À1 root fresh weight, and serially diluted, or pieces of 1 cm length were used directly for inoculation before or after surface sterilization.…”

“…have interesting features with respect to plant± microbe interactions. They have been isolated from the interior of roots and culms of Kallar grass (Reinhold et al, 1986;Reinhold-Hurek et al, 1993a), a pioneer plant on salt-affected, often¯ooded, low-fertility soils in the Punjab of Pakistan. Their main entry points into roots are near the root tips at the zone of elongation and differentiation, and at the emergence points of lateral roots, as has also been described for Acetobacter diazotrophicus and H. seropedicae (James et al, 1997;James and Olivares, 1998).…”

The oil‐contaminated soil diazotroph Azoarcus oleariusDQS‐4T is genetically and phenotypically similar to the model grass endophyte Azoarcus sp. BH72

“…Azoarcus was first discovered after several strains were isolated from surface sterilized roots of the halotolerant C4 Poaceae species Leptochloa fusca L. (kallar grass) in Pakistan (Reinhold-Hurek et al, 1986;Reinhold et al, 1987). The new species Azoarcus communis and A. indigens were subsequently described (Reinhold-Hurek et al, 1993), but the most effective PGPR Azoarcus strain isolated from Pakistani kallar grass was BH72.…”

Novel insights into ecological distribution and plant growth promotion by nitrogen‐fixing endophytes – how specialised are they?

“…Several endophytic diazotrophic bacteria have become model species for characterisation of interactions with gramineous crops and for analysing the infection and colonisation process, including Azoarcus sp., Herbaspirillum seropedicae, Azospirillum brasilense, Gluconacetobacter diazotrophicus, Klebsiella pneumoniae 342 and Burkholderia spp (Reinhold-Hurek and Hurek, 2011;Carvalho et al, 2014Carvalho et al, , 2016Do Amaral et al, 2016). However, a strain of Azoarcus sp., designated as BH72, which was originally isolated from Kallar grass in the Punjab region of Pakistan (Reinhold et al, 1986;Hurek et al, 1987), stands out as being the most highly studied diazotrophic endophyte. The genome sequence of Azoarcus sp.…”