NodC-based evaluation of the occurrence of bacteria in nodules of Pisum sativum isolated on YEM agar

Anna Lenart-Boroń; Tadeusz Zając; Piotr Mateusz Boroń; Agnieszka Klimek-Kopyra

doi:10.2478/boku-2019-0005

Figures & Tables

BamHI digestion of nodC gene of 17 selected Rhizobium leguminosarum samples. Letters at the top of the figure show experimental variants, whereas letters at the bottom of the figure show the digestion patterns.Legend for the top of the figure: Ku, uninoculated “Klif ” pea plants; KI, “Klif ” plants inoculated with IUNG; KN, “Klif ” plants inoculated with Nitragine™; Tu, uninoculated “Tarchalska” pea plants; TI, “Tarchalska” plants inoculated with IUNG; TN, “Tarchalska” plants inoculated with Nitragine™; IUNG, bacterial isolates derived from the IUNG inoculant; LR, LC, SR, and SC refer to the types of nodules: LR, large red; LC, large cream; SR, small red; SC, small cream.Legend for the bottom of the figure: M, GeneRuler™ 1kb DNA Ladder (Thermo Scientific); A, B, and C, clear digestion profiles; A + B, mixed digestion profiles.Abbildung 1. BamHI-Verdauung des nodC-Gens von 17 ausgewählten Proben von Rhizobium leguminosarum. Buchstaben oben in der Abbildung zeigen experimentelle Varianten, während Buchstaben unten in der Abbildung die Verdauungsmuster zeigen.

Detection of nodC gene in the studied bacterial isolatesTabelle 4_ Nachweis des nodC-Gens in den untersuchten Bakterienisolaten

Isolate No.	Variant	Nodule	Number of isolates per variant	nodC presence
1	Klif (uninoculated)	Large red	1	Yes
2	Klif (uninoculated)	Large red	2	No
3	Klif (uninoculated)	Small red	1	No
4	Klif (uninoculated)	Small red	2	No
5	Klif (uninoculated)	Large cream	1	Yes
6	Klif (uninoculated)	Small cream	1	Yes
7	Klif (uninoculated)	Small cream	2	No
8	Tarchalska (uninoculated)	Large red	1	Yes
9	Tarchalska (uninoculated)	Small red	1	Yes
10	Tarchalska (uninoculated)	Small red	2	No
11	Tarchalska (uninoculated)	Large cream	1	Yes
12	Tarchalska (uninoculated)	Small cream	2	Yes
13	Klif + IUNG	Large red	1	Yes
14	Klif + IUNG	Small red	1	Yes
15	Klif + IUNG	Large cream	1	Yes
16	Klif + IUNG	Small cream	1	No
17	Tarchalska + IUNG	Large red	1	Yes
18	Tarchalska + IUNG	Large red	2	No
19	Tarchalska + IUNG	Large red	3	No
20	Tarchalska + IUNG	Large red	4	No
21	Tarchalska + IUNG	Small red	1	No
22	Tarchalska + IUNG	Small red	2	No
23	Tarchalska + IUNG	Small red	3	No
24	Tarchalska/IUNG	Large cream	1	No
25	Tarchalska + IUNG	Large cream	2	No
26	Tarchalska + IUNG	Large cream	3	No
27	Tarchalska + IUNG	Small cream	1	No
28	Klif + Nitragine™	Large red	1	No
29	Klif + Nitragine™	Large red	2	No
30	Klif + Nitragine™	Large red	3	No
31	Klif + Nitragine™	Large red	4	No
32	Klif + Nitragine™	Small red	1	Yes
33	Klif + Nitragine™	Small red	2	No
34	Klif + Nitragine™	Large cream	1	Yes
35	Klif + Nitragine™	Small cream	1	Yes
36	Tarchalska/Nitragine™	Large red	1	Yes
37	Tarchalska + Nitragine™	Large red	2	No
38	Tarchalska + Nitragine™	Small red	1	Yes
39	Tarchalska + Nitragine™	Large cream	1	Yes
40	Tarchalska + Nitragine™	Small cream	1	Yes
41	Tarchalska + Nitragine™	Small cream	2	Yes
42	Tarchalska/Nitragine™	Small cream	3	Yes
43	Tarchalska + Nitragine™	Small cream	4	Yes
44	Nitragine™	Nonapplicable	1	No
45	IUNG	Nonapplicable	1	Yes
46	R. leguminosarum bv. viceae DSM 30143	Nonapplicable	1	No

Oligonucleotides used for nodC amplificationTabelle 1_ Für die nodC-Amplifikation verwendete Oligonukleotide

Primer	5^′–3^′ nucleotide sequence
nodCF	AYGTHGTYGAYGACGGTTC
nodCFu	AYGTHGTYGAYGACGGITC
nodCF2	AYGTHGTYGAYGACGGCTC
nodCF4	AYGTHGTYGAYGACGGATC
nodCFn	AGGTGGTYGAYGACGGTTC
nodCI	CGYGACAGCCANTCKCTATTG

Identification of bacteria colonizing pea nodulesTabelle 3_ Identifizierung der Bakterien, welche die Erbsenknöllchen besiedelten

Variant	Bacteria species	Maximum identity (%)
IUNG	Rhizobium leguminosarum bv. viciae USDA 2370 strain USDA 2370; ATCC 10004	99
Nitragine^TM	Achromobacter insolitus strain LMG 6003	99
Klif (uninoculated)	Bacillus megaterium strain IAM 13418	99
Tarchalska (uninoculated)	Stenotrophomonas maltophilia strain IAM 12423	99
Tarchalska + IUNG	Stenotrophomonas maltophilia strain IAM 12423	99
Klif + Nitragine^TM	Stenotrophomonas maltophilia strain IAM 12423	98
Tarchalska + Nitragine^TM	Stenotrophomonas maltophilia strain IAM 12423	99

The mean numbers of colony forming units (CFUs) of bacteria colonizing pea nodules per one nodule depending on variants (pea varieties and inoculants)_Tabelle 2_ Die Durchschnittswerte koloniebildender Einheiten (KBE) von Bakterien, die Erbsenknollen besiedeln, pro Knöllchen in Abhängigkeit der Varianten (Erbsensorten und Inokulen)_

Variant	Nodules
	Large cream	Small cream	Large red	Small red	Mean
Klif (uninoculated)	20.5	87.0	128.5	52.0	72.0^b
Tarchalska (uninoculated)	6.0	110.0	157.5	36.0	77.4^b
Klif + IUNG	84.0	97.5	310.0	212.0	175.9^a
Tarchalska + IUNG	110.0	45.0	101.0	49.0	76.3^b
Klif + Nitragine^TM	54.0	79.5	67.0	39.0	59.9^b
Tarchalska + Nitragine^TM	45.0	54.0	32.0	53.0	46.0^b
Mean	53.2^b	78.8^ab	132.7^a	73.5^ab

NodC-based evaluation of the occurrence of bacteria in nodules of Pisum sativum isolated on YEM agar

Figures & Tables

Figure 1

Detection of nodC gene in the studied bacterial isolatesTabelle 4_ Nachweis des nodC-Gens in den untersuchten Bakterienisolaten

Oligonucleotides used for nodC amplificationTabelle 1_ Für die nodC-Amplifikation verwendete Oligonukleotide

Identification of bacteria colonizing pea nodulesTabelle 3_ Identifizierung der Bakterien, welche die Erbsenknöllchen besiedelten

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