Mixed bacterial fermentation can control the growth and development of Verticillium dahliae

Figures & data

Table 1. Primers used in this study.

Gene	Primer sequence 5′–3′	Tm (°C)
NADPH oxidase	5′ GGCTCCTTTGAGTGGTTCCG 3′ 5′ TTCGTTGGCGACCTCAGACC 3′	48
Glutathione reductase	5′ AACATTCCCTCGGTCGTCTT 3′ 5′ TGGCGGTGAACTTGGTCTT 3′	48
Glycerol-3-phosphate dehydrogenase	5′ TCCACCGCCCCTACTTCCAT 3′ 5′ CAGCCTTGGCGTTGTCACCC 3′	48
Chitinase	5′ ATACGGGCGAGACTACCAC 3′ 5′ CTTCTCCAGGTCGGCATAG 3′	48
VMK1	5′ GCCGACGATGGAGGACTACT 3′ 5′ GCTCAGCGTGTCCTTGTGC 3′	48
VDgarp	5′ GAGCGGCTGCTTTGGGTTT 3′ 5′ AGTTGATGGGTGCTGCTTGTAGAC 3′	48
β-tubulin	5′ GGCCGCCTCTGACTTCCGTAAC 3′ 5′ CTCGACCCCTTCATGGCAACCTT 3′	48

β-tubulin was a reference gene; VMK1 and GARP are related to mycelium development [21]; G1-3 is glycerol-3-phosphate dehydrogenase, related to cell infiltration; NADPH is an oxidase gene, GLU encodes glutathione reductase; these two genes are involved in the oxidative response; CHIT encodes chitinase, it can induce the synthesis of cell wall hydrolytic enzymes in CWI pathway and cause the cell death of Verticillium dahliae.

Table 2. Inhibitory effects of cell-free mixed culture filtrate on conidia production of V. dahliae.

No.	Colony size	Conidia number (CFU 10^6）	⊿1(%)	No.	Colony size	Conidia number (CFU 10^6)	⊿1(%)
CK	7.37 ± 0.12	9.07 ± 0.15	–	A150	4.57 ± 0.12	5.53 ± 0.31	39
M10	0.83 ± 0.12	0.2 ± 0.1	97.8	A1100	5.83 ± 0.25	8.07 ± 0.15	11
M50	3.1 ± 0.2	2.9 ± 0.36	68	C-910	1.53 ± 0.15	2.63 ± 0.21	71
M100	4.9 ± 0.2	5.77 ± 0.35	36.4	C-950	4.2 ± 0.1	5.07 ± 0.32	44.1
A110	2.03 ± 0.12	3.23 ± 0.31	64.4	C-9100	5.63 ± 0.25	7.73 ± 0.4	14.8

M₁₀, M₅₀ and M₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of mixed fermentation culture filtrate (A1: c-9 = 1:9), respectively; A1₁₀, A1₅₀ and A1₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of Sphingobacterium A1 fermentation filtrate respectively; C-9₁₀, C-9₅₀ and C-9₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of B. tequilensis C-9 fermentation filtrate, respectively.

Table 3. Inhibitory effects of the mixed-culture filtrate on the germination of V. dahliae spores.

No.	Germination rates (%)	⊿2(%)	No.	Germination rates (%)	⊿2 (%)
CK	63.33 ± 1.53	–	A150	21.67 ± 1.15	65.8
M10	0 ± 0	100	A1100	31.33 ± 2.08	50.5
M50	0 ± 0	100	C-910	0 ± 0	100
M100	10.67 ± 0.58	83.2	C-950	0 ± 0	100
A110	8.33 ± 0.58	86.8	C-9100	18 ± 1.73	14.8

M₁₀, M₅₀ and M₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of mixed fermentation culture filtrate (A1: c-9 = 1:9), respectively; A1₁₀, A1₅₀ and A1₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of Sphingobacterium A1 fermentation filtrate respectively; C-9₁₀, C-9₅₀ and C-9₁₀₀ denote 1:10, 1:50 and 1:100 dilutions of B. tequilensis C-9 fermentation filtrate, respectively.

Figure 1. Effects of mixed-culture filtrates on Verticillium dahliae protein secretion.

Note: The fermentation broth obtained by the mixed culture of A1 and C-9 was filtered through a microporous filtration membrane. After mixing the filtrate with PDA medium, the protein content of the inhibited Verticillium culture was measured in the plate.

Figure 2. Effects of the mixed-culture filtrate against Verticillium dahliae mycelia observed under a microscope.

Note: A.CK: Morphology of hyphae uninhibited by fermentation broth; B.A1: Morphology of hyphae inhibited by A1 monoculture fermentation filtrate; C.C-9: Morphology of hyphae inhibited by C-9 monoculture fermentation filtrate; D.M: Morphology of hyphae inhibited by the mixed-culture fermentation filtrate of A-1 and C-9.

Figure 3. Effects of mixed culture filtrates on the gene expression of V. dahliae. VMK1 (A) and GARP (B); G1-3 (C); GLU (D), NADPH oxidase (E), CHIT (F). β-tubulin was used as internal control.

Table 4. Application of microbial agents promotes cotton growth.

Parcel of land	Variety	Plant number	Plant height (cm)	Number of buds	Number of bolls	Lint weight (g)	Increase (%)
Mildly affected parcel	Xin Lu Zao 7CK	40	76.05 ± 3.73	10.25 ± 3.65	11.4 ± 2.34	3.88 ± 0.08	–
	Xin Lu Zao 7TR	40	80.73 ± 4.48	11.53 ± 3.52	13.33 ± 3.51	4.34 ± 0.09	11.86
	Xin Lu Zao 33CK	40	73.63 ± 4.93	10.68 ± 5.01	15.25 ± 3.28	3.43 ± 0.09	–
	Xin Lu Zao 33TR	40	81.85 ± 5.03	13.38 ± 3.29	15.7 ± 2.99	3.93 ± 0.04	14.58
Severely affected parcel	Xin Lu Zao 7CK	40	75.6 ± 3.41	9.93 ± 2.97	12.95 ± 4.33	3 ± 0.06	–
	Xin Lu Zao 7TR	40	80.53 ± 4.49	10.98 ± 2.8	16.75 ± 2.99	3.53 ± 0.12	17.67
	Xin Lu Zao 33CK	40	72.8 ± 4.24	9.98 ± 4.32	12.6 ± 3.34	2.58 ± 0.05	–
	Xin Lu Zao 33TR	40	79.98 ± 3.68	12.7 ± 3.81	14.15 ± 2.56	2.98 ± 0.12	15.5

Xin Lu Zao 7: cotton variety susceptible to Verticillium wilt.

Xin Lu Zao 33: cotton variety resistant to verticillium wilt.

CK, control (treated with water).

TR, treatment group (treated with microbial agent).

*Microbial agent refers to the compound microbial fungicide developed by adding the corresponding carrier and auxiliary agent to the mixed fermentation broth). It was applied with water droplets (twice at seedling stage and bud stage of cotton, respectively) at a dose of 20 g/m².

Figure 4. Effects of microbial agents on Verticillium wilt disease of cotton in the budding stage of nurseries. X7, Xin Lu Zao 7 (susceptible); X33, Xin Lu Zao 33 (resistant).

Figure 5. Effects of microbial agents on the occurrence of cotton Verticillium wilt in the bolling period in the mildly affected parcel (A, B) and in the severely affected parcel (C, D).

Figure 6. Incidence grades of Verticillium wilt in cotton plants at different stages and areas: bud stage in heavy disease areas (A, B), boll stage in light-disease areas (C, D), ball stage in heavy disease areas (E, F).

Note: X7CK: Xin Lu Zao 7 without treatment; X7TR: Xin Lu Zao 7 with treatment; X33CK: Xin Lu Zao 33 without treatment; X33TR: Xin Lu Zao 33 with treatment. Incidence Grades (1) yellowing or withering leaves less than 25%; (2) yellowing or withering leaves 25–50%; (3) yellowing or withered leaves ranging from 50% to 75%; (4) yellowish withered leaves greater than 75%.

Liang M. Screening of abnormal nuclear development mutants and identification of related genes in Rotadella dahlia. Nanjing (China): Nanjing Normal University; 2015.

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