微生物学报

Solutions	PM1, 2A	PM3B, 5, 6,7, 8	PM4A	PM9, 10
FF-IF inoculum (mL)	20.00	50.00	10.00	20.00
d-glucose (mL)	-	1.88	0.38	0.75
PM additive solution (mL)	-	5.00	-	2.00
Mycelial fragment suspension (mL)	0.50	1.25	0.25	0.50
Sterile water (mL)	3.50	1.88	1.38	0.75
Total volume (mL)	24.00	60.00	12.00	24.00

Solutions	PM1, 2A	PM3B, 5, 6,7, 8	PM4A	PM9, 10
FF-IF inoculum (mL)	20.00	50.00	10.00	20.00
d-glucose (mL)	-	1.88	0.38	0.75
PM additive solution (mL)	-	5.00	-	2.00
Mycelial fragment suspension (mL)	0.50	1.25	0.25	0.50
Sterile water (mL)	3.50	1.88	1.38	0.75
Total volume (mL)	24.00	60.00	12.00	24.00

Microplate	Well	Substrate	Sclerotial formation
PM1	B02	d-sorbitol	++
B03	Glycerol	+
B08	d-xylose	++
C03	d,l-malic acid	+
C08	Acetic acid	+
D05	Tween-40	+
D10	Lactulose	+
E07	α-hydroxybutyric acid	+
F05	Fumaric acid	+
H06	l-lyxose	+
PM2A	B01	N-acetyl-d-galactosamine	++
B05	d-arabinose	++
C01	Gentiobiose	+
C04	d-melezitose	++
C12	Palatinose	+
D01	d-raffinose	+
D04	l-sorbose	+
D07	Turanose	+
G12	l-methionine	+

Microplate	Well	Substrate	Sclerotial formation
PM1	B02	d-sorbitol	++
B03	Glycerol	+
B08	d-xylose	++
C03	d,l-malic acid	+
C08	Acetic acid	+
D05	Tween-40	+
D10	Lactulose	+
E07	α-hydroxybutyric acid	+
F05	Fumaric acid	+
H06	l-lyxose	+
PM2A	B01	N-acetyl-d-galactosamine	++
B05	d-arabinose	++
C01	Gentiobiose	+
C04	d-melezitose	++
C12	Palatinose	+
D01	d-raffinose	+
D04	l-sorbose	+
D07	Turanose	+
G12	l-methionine	+

Well	Substrate	Sclerotial formation
A04	Nitrate	+
A10	l-aspartic acid	++
A12	l-glutamic acid	+
B01	l-glutamine	++
B10	l-serine	+
C01	l-tyrosine	+
C05	d-aspartic acid	+
C08	d-serine	+
D04	Hydroxylamine	+
D06	N-amylamine	+
D12	Agmatine	+
E08	d-glucosamine	+
E11	N-acetyl-d-glucosamine	++++
F03	Adenosine	+++
F04	Cytidine	+
F08	Thymine	+
F09	Thymidine	+++
H03	Ala-Glu	+
H04	Ala-Gly	+
H05	Ala-His	+
H10	Gly-Glu	++

Well	Substrate	Sclerotial formation
A04	Nitrate	+
A10	l-aspartic acid	++
A12	l-glutamic acid	+
B01	l-glutamine	++
B10	l-serine	+
C01	l-tyrosine	+
C05	d-aspartic acid	+
C08	d-serine	+
D04	Hydroxylamine	+
D06	N-amylamine	+
D12	Agmatine	+
E08	d-glucosamine	+
E11	N-acetyl-d-glucosamine	++++
F03	Adenosine	+++
F04	Cytidine	+
F08	Thymine	+
F09	Thymidine	+++
H03	Ala-Glu	+
H04	Ala-Gly	+
H05	Ala-His	+
H10	Gly-Glu	++

Well	Substrate	Sclerotial formation
A09	Adenosine 3′-monophosphate	+
B09	Guanosine 3′-monophosphate	+
C04	d-glucose-6-phosphate	++
D04	Phospho-l-arginine	+
D07	o-phospho-l-threonine	+
D09	Uridine 3′-monophosphate	++++
E04	Phosphoryl choline	++++
E11	Inositol hexaphosphate	+
F06	Dithiophosphate	+
G12	l-methionine sulfone	+
H02	Thiourea	+
H03	1-thio-β-d-glucose	+
H05	Taurocholic acid	+
H06	Taurine	+
H09	Butane sulfonic acid	+
H11	Methane sulfonic acid	+

Well	Substrate	Sclerotial formation
A09	Adenosine 3′-monophosphate	+
B09	Guanosine 3′-monophosphate	+
C04	d-glucose-6-phosphate	++
D04	Phospho-l-arginine	+
D07	o-phospho-l-threonine	+
D09	Uridine 3′-monophosphate	++++
E04	Phosphoryl choline	++++
E11	Inositol hexaphosphate	+
F06	Dithiophosphate	+
G12	l-methionine sulfone	+
H02	Thiourea	+
H03	1-thio-β-d-glucose	+
H05	Taurocholic acid	+
H06	Taurine	+
H09	Butane sulfonic acid	+
H11	Methane sulfonic acid	+

Well	Substrate	Sclerotial formation	Well	Substrate	Sclerotial formation
A06	l-aspartic acid	+	E07	β-alanine	+
A08	l-glutamic acid	+	E08	d-pantothenic acid	+
A10	Adenine	+	E09	Orotic acid	+
A11	Adenosine	++	E11	Uridine	+
A12	2′-deoxyadenosine	+	E12	2′-deoxyuridine	+
B01	l-glutamine	+	F01	Quinolinic acid	+
B02	Glycine	+	F02	Nicotinic acid	+
B04	l-isoleucine	+	F03	Nicotinamide	++
B06	l-lysine	+	F04	β-nicotinamide adenine dinucleotide	+
B08	l-phenylalanine	+	F05	δ-amino-levulinic acid	+
B09	Guanosine 3′,5′-cyclic monophosphate	+	F06	Hematin	+
B10	Guanine	+	F07	Deferoxamine	+
B11	Guanosine	+	F08	α-d-glucose	+
B12	2′-deoxyguanosine	+	F09	N-acetyl-d-glucosamine	+
C01	l-proline	+	F10	Thymine	+
C03	l-threonine	+	F11	Glutathione	+
C06	l-valine	+	F12	Thymidine	+
C07	l-isoleucine+l-valine	+	G01	Oxaloacetic acid	+
C08	Hydroxy-l-proline	++	G03	Cyanocobalamin	+
C09	(5)4-amino-imidazole-4(5)-carboxamide	+	G04	ρ-amino-benzoic acid	+
C10	Hypoxanthine	+	G05	Folic acid	+
C11	Inosine	+	G06	Inosine+thiamine	+
C12	2′-deoxyinosine	+	G08	Thiamine pyrophosphate	+
D02	l-citrulline	+	G10	Pyrrolo-quinoline quinone	+
D03	Chorismic acid	+	G12	Myo-inositol	++
D04	(-)Shikimic acid	+	H01	Butyric acid	+
D05	l-homoserine lactone	+	H03	α-ketobutyric acid	+
D07	d-aspartic acid	++	H04	Caprylic acid	+
D08	d-glutamic acid	+	H06	d,l-mevalonic acid	+
D10	Cytosine	+	H07	d,l-carnitine	+
D11	Cytidine	+	H08	Choline	++
D12	2′-deoxycytidine	+	H10	Tween-40	+
E01	Putrescine	+	H11	Tween-60	+
E02	Spermidine	+	H12	Tween-80	+++
E03	Spermine	+

Well	Substrate	Sclerotial formation	Well	Substrate	Sclerotial formation
A06	l-aspartic acid	+	E07	β-alanine	+
A08	l-glutamic acid	+	E08	d-pantothenic acid	+
A10	Adenine	+	E09	Orotic acid	+
A11	Adenosine	++	E11	Uridine	+
A12	2′-deoxyadenosine	+	E12	2′-deoxyuridine	+
B01	l-glutamine	+	F01	Quinolinic acid	+
B02	Glycine	+	F02	Nicotinic acid	+
B04	l-isoleucine	+	F03	Nicotinamide	++
B06	l-lysine	+	F04	β-nicotinamide adenine dinucleotide	+
B08	l-phenylalanine	+	F05	δ-amino-levulinic acid	+
B09	Guanosine 3′,5′-cyclic monophosphate	+	F06	Hematin	+
B10	Guanine	+	F07	Deferoxamine	+
B11	Guanosine	+	F08	α-d-glucose	+
B12	2′-deoxyguanosine	+	F09	N-acetyl-d-glucosamine	+
C01	l-proline	+	F10	Thymine	+
C03	l-threonine	+	F11	Glutathione	+
C06	l-valine	+	F12	Thymidine	+
C07	l-isoleucine+l-valine	+	G01	Oxaloacetic acid	+
C08	Hydroxy-l-proline	++	G03	Cyanocobalamin	+
C09	(5)4-amino-imidazole-4(5)-carboxamide	+	G04	ρ-amino-benzoic acid	+
C10	Hypoxanthine	+	G05	Folic acid	+
C11	Inosine	+	G06	Inosine+thiamine	+
C12	2′-deoxyinosine	+	G08	Thiamine pyrophosphate	+
D02	l-citrulline	+	G10	Pyrrolo-quinoline quinone	+
D03	Chorismic acid	+	G12	Myo-inositol	++
D04	(-)Shikimic acid	+	H01	Butyric acid	+
D05	l-homoserine lactone	+	H03	α-ketobutyric acid	+
D07	d-aspartic acid	++	H04	Caprylic acid	+
D08	d-glutamic acid	+	H06	d,l-mevalonic acid	+
D10	Cytosine	+	H07	d,l-carnitine	+
D11	Cytidine	+	H08	Choline	++
D12	2′-deoxycytidine	+	H10	Tween-40	+
E01	Putrescine	+	H11	Tween-60	+
E02	Spermidine	+	H12	Tween-80	+++
E03	Spermine	+

Microplate	Well	Substrate	Sclerotial formation	Microplate	Well	Substrate	Sclerotial formation
PM6	A05	Ala-Asn	+	PM7	D01	Phe-Pro	+++
A06	Ala-Glu	+	D10	Pro-Phe	++++
A09	Ala-Leu	+++	E04	Ser-Leu	+
A11	Ala-Phe	+	E06	Ser-Phe	+
B01	Ala-Ser	+	F04	Thr-Met	+
B02	Ala-Thr	+	F06	Trp-Ala	+
B05	Arg-Ala	+	G02	Trp-Ser	+
B07	Arg-Asp	++	H03	Val-Arg	++
B08	Arg-Gln	+	H04	Val-Asn	+
C02	Arg-Phe	++	H10	Val-Tyr	++++
C04	Arg-Trp	++++	PM8	A03	Ala-Asp	++
C07	Asn-Glu	+	A07	Ala-Val	+++
D05	Gln-Gln	+	A10	Asp-Gly	+
D06	Gln-Gly	++	A11	Glu-Ala	+
D07	Glu-Asp	+	A12	Gly-Asn	+
D08	Glu-Glu	+	B01	Gly-Asp	++
D09	Glu-Gly	+	B02	Gly-Ile	+
D10	Glu-Ser	+	C11	Pro-Arg	+
E01	Glu-Val	+	D07	Ser-Asn	+
E04	Gly-Cys	+	D08	Ser-Asp	+
E11	Gly-Pro	+	D10	Ser-Glu	+
F07	His-Leu	+	E02	Thr-Ser	++
G04	Ile-Arg	+	E08	Val-Glu	+
G05	Ile-Gln	+	E10	Val-Met	++++
G10	Ile-Phe	+++	F07	d-Ala-d-Ala	+
H02	Ile-Tyr	+++	G01	γ-Glu-Gly	+++
PM7	A02	l-glutamine	+	G03	Gly-d-Ala	++
B04	Lys-Trp	+	G04	Gly-d-Asp	+
B07	Met-Arg	++++	H04	Gly-Gly-Ile	+
C03	Met-Lys	+	H12	Tyr-Gly-Gly	+
C09	Phe-Ala	+

Microplate	Well	Substrate	Sclerotial formation	Microplate	Well	Substrate	Sclerotial formation
PM6	A05	Ala-Asn	+	PM7	D01	Phe-Pro	+++
A06	Ala-Glu	+	D10	Pro-Phe	++++
A09	Ala-Leu	+++	E04	Ser-Leu	+
A11	Ala-Phe	+	E06	Ser-Phe	+
B01	Ala-Ser	+	F04	Thr-Met	+
B02	Ala-Thr	+	F06	Trp-Ala	+
B05	Arg-Ala	+	G02	Trp-Ser	+
B07	Arg-Asp	++	H03	Val-Arg	++
B08	Arg-Gln	+	H04	Val-Asn	+
C02	Arg-Phe	++	H10	Val-Tyr	++++
C04	Arg-Trp	++++	PM8	A03	Ala-Asp	++
C07	Asn-Glu	+	A07	Ala-Val	+++
D05	Gln-Gln	+	A10	Asp-Gly	+
D06	Gln-Gly	++	A11	Glu-Ala	+
D07	Glu-Asp	+	A12	Gly-Asn	+
D08	Glu-Glu	+	B01	Gly-Asp	++
D09	Glu-Gly	+	B02	Gly-Ile	+
D10	Glu-Ser	+	C11	Pro-Arg	+
E01	Glu-Val	+	D07	Ser-Asn	+
E04	Gly-Cys	+	D08	Ser-Asp	+
E11	Gly-Pro	+	D10	Ser-Glu	+
F07	His-Leu	+	E02	Thr-Ser	++
G04	Ile-Arg	+	E08	Val-Glu	+
G05	Ile-Gln	+	E10	Val-Met	++++
G10	Ile-Phe	+++	F07	d-Ala-d-Ala	+
H02	Ile-Tyr	+++	G01	γ-Glu-Gly	+++
PM7	A02	l-glutamine	+	G03	Gly-d-Ala	++
B04	Lys-Trp	+	G04	Gly-d-Asp	+
B07	Met-Arg	++++	H04	Gly-Gly-Ile	+
C03	Met-Lys	+	H12	Tyr-Gly-Gly	+
C09	Phe-Ala	+

Well	Substrate	Sclerotialformation
D01	3% Potassium chloride	+
D02	4% Potassium chloride	+
D05	2% Sodium sulfate	+
D06	3% Sodium sulfate	++
D07	4% Sodium sulfate	+
D08	5% Sodium sulfate	++
D09	5% Ethylene glycol	+
D10	10% Ethylene glycol	++
D11	15% Ethylene glycol	+
D12	20% Ethylene glycol	++
E07	2% Urea	+
F01	1% Sodium lactate	++
F02	2% Sodium lactate	++
F11	11% Sodium lactate	+
G01	20 mmol/L Sodium phosphate pH 7.0	++
G02	50 mmol/L Sodium phosphate pH 7.0	+
G04	200 mmol/L Sodium phosphate pH 7.0	++
G05	20 mmol/L Sodium benzoate pH 5.2	++
G09	10 mmol/L Ammonium sulfate pH 8.0	++++
G10	20 mmol/L Ammonium sulfate pH 8.0	++++
G11	50 mmol/L Ammonium sulfate pH 8.0	++
G12	100 mmol/L Ammonium sulfate pH 8.0	++
H01	10 mmol/L Sodium nitrate	++
H02	20 mmol/L Sodium nitrate	+
H03	40 mmol/L Sodium nitrate	++
H04	60 mmol/L Sodium nitrate	+++
H05	80 mmol/L Sodium nitrate	++
H06	100 mmol/L Sodium nitrate	++

Well	Substrate	Sclerotialformation
D01	3% Potassium chloride	+
D02	4% Potassium chloride	+
D05	2% Sodium sulfate	+
D06	3% Sodium sulfate	++
D07	4% Sodium sulfate	+
D08	5% Sodium sulfate	++
D09	5% Ethylene glycol	+
D10	10% Ethylene glycol	++
D11	15% Ethylene glycol	+
D12	20% Ethylene glycol	++
E07	2% Urea	+
F01	1% Sodium lactate	++
F02	2% Sodium lactate	++
F11	11% Sodium lactate	+
G01	20 mmol/L Sodium phosphate pH 7.0	++
G02	50 mmol/L Sodium phosphate pH 7.0	+
G04	200 mmol/L Sodium phosphate pH 7.0	++
G05	20 mmol/L Sodium benzoate pH 5.2	++
G09	10 mmol/L Ammonium sulfate pH 8.0	++++
G10	20 mmol/L Ammonium sulfate pH 8.0	++++
G11	50 mmol/L Ammonium sulfate pH 8.0	++
G12	100 mmol/L Ammonium sulfate pH 8.0	++
H01	10 mmol/L Sodium nitrate	++
H02	20 mmol/L Sodium nitrate	+
H03	40 mmol/L Sodium nitrate	++
H04	60 mmol/L Sodium nitrate	+++
H05	80 mmol/L Sodium nitrate	++
H06	100 mmol/L Sodium nitrate	++

Well	Substrate	Sclerotial formation
A02	pH 4.0	+++
A03	pH 4.5	+++
A04	pH 5.0	++
A05	pH 5.5	++
A06	pH 6.0	+
A07	pH 7.0	+
A10	pH 9.0	++
A11	pH 9.5	+++
A12	pH 10.0	+++
B01	pH 4.5	+++
B02	pH 4.5+l-alanine	+
B03	pH 4.5+l-arginine	++
B04	pH 4.5+l-asparagine	++
B06	pH 4.5+l-glutamic acid	+
B07	pH 4.5+l-glutamine	+++
B08	pH 4.5+Glycine	+
B09	pH 4.5+l-histidine	+
C02	pH 4.5+l-phenylalanine	++
C03	pH 4.5+l-proline	++++
C04	pH 4.5+l-serine	++
C05	pH 4.5+l-threonine	+
C07	pH 4.5+l-citrulline	++
C08	pH 4.5+l-valine	+++
C09	pH 4.5+Hydroxy-l-proline	++
C10	pH 4.5+l-ornithine	++
C11	pH 4.5+l-homoarginine	++
C12	pH 4.5+l-homoserine	+
D02	pH 4.5+l-norleucine	++
D11	pH 4.5+Trimethylamine-N-oxide	+
E10	pH 9.5+l-isoleucine	++
F12	pH 9.5+l-homoserine	+
G03	pH 9.5+l-norvaline	++
H01	X-caprylate	+++
H05	X-β-d-galactoside	+
H06	X-α-d-glucuronide	+
H07	X-β-d-glucuronide	++
H08	X-β-d-glucosaminide	++
H09	X-β-d-galactosaminide	++
H10	X-α-d-mannoside	++
H12	X-SO₄	++

Well	Substrate	Sclerotial formation
A02	pH 4.0	+++
A03	pH 4.5	+++
A04	pH 5.0	++
A05	pH 5.5	++
A06	pH 6.0	+
A07	pH 7.0	+
A10	pH 9.0	++
A11	pH 9.5	+++
A12	pH 10.0	+++
B01	pH 4.5	+++
B02	pH 4.5+l-alanine	+
B03	pH 4.5+l-arginine	++
B04	pH 4.5+l-asparagine	++
B06	pH 4.5+l-glutamic acid	+
B07	pH 4.5+l-glutamine	+++
B08	pH 4.5+Glycine	+
B09	pH 4.5+l-histidine	+
C02	pH 4.5+l-phenylalanine	++
C03	pH 4.5+l-proline	++++
C04	pH 4.5+l-serine	++
C05	pH 4.5+l-threonine	+
C07	pH 4.5+l-citrulline	++
C08	pH 4.5+l-valine	+++
C09	pH 4.5+Hydroxy-l-proline	++
C10	pH 4.5+l-ornithine	++
C11	pH 4.5+l-homoarginine	++
C12	pH 4.5+l-homoserine	+
D02	pH 4.5+l-norleucine	++
D11	pH 4.5+Trimethylamine-N-oxide	+
E10	pH 9.5+l-isoleucine	++
F12	pH 9.5+l-homoserine	+
G03	pH 9.5+l-norvaline	++
H01	X-caprylate	+++
H05	X-β-d-galactoside	+
H06	X-α-d-glucuronide	+
H07	X-β-d-glucuronide	++
H08	X-β-d-glucosaminide	++
H09	X-β-d-galactosaminide	++
H10	X-α-d-mannoside	++
H12	X-SO₄	++

基于Biolog表型芯片的立枯丝核菌菌核形成所需代谢底物分析

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向立刚 ¹^,² , 汪汉成 ¹ , 蔡刘体 ¹ , 陈丽莉 ¹ , 张文建 ² , 王军 ² , 孟建玉 ¹ , 杨靓 ³ , 温明霞 ²

微生物学报 | 研究报告 2025,65(5): 2190-2200

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微生物学报 | 研究报告 2025, 65(5): 2190-2200

基于Biolog表型芯片的立枯丝核菌菌核形成所需代谢底物分析

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向立刚¹^,², 汪汉成¹, 蔡刘体¹, 陈丽莉¹, 张文建², 王军², 孟建玉¹, 杨靓³, 温明霞²

作者信息

^1.贵州省烟草科学研究院，贵州贵阳

^2.贵州省烟草公司遵义市公司，贵州遵义

^3.福建农林大学，植物病毒研究所，福建福州

Biolog phenotypic microarray reveals the metabolic substrates required for sclerotial formation of Rhizoctonia solani

Ligang XIANG¹^,², Hancheng WANG¹, Liuti CAI¹, Lili CHEN¹, Wenjian ZHANG², Jun WANG², Jianyu MENG¹, Liang YANG³, Mingxia WEN²

Affiliations

^1.Guizhou Academy of Tobacco Science, Guiyang, Guizhou, China

^2.Guizhou Tobacco Company Zunyi Company, Zunyi, Guizhou, China

^3.Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China

出版时间: 2025-05-04 doi: 10.13343/j.cnki.wsxb.20240778

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摘要

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【目的】分析立枯丝核菌菌核形成所需的代谢底物，旨在了解营养元素和环境因子对其菌核形成的影响规律。【方法】基于Biolog表型芯片技术测定663种营养物质、96种渗透压和96种pH环境下立枯丝核菌的菌核形成能力。【结果】在供试的营养物质及环境条件中，分别有19/95的碳源、21/95的氮源、16/94的磷硫源、69/94的营养补充物、61/282的肽类氮源、28/96的渗透压环境以及40/96的pH环境可以诱导立枯丝核菌形成菌核；其中，N-乙酰-d-氨基葡萄糖、尿苷3′-单磷酸盐、磷酸胆碱和5种二肽(Arg-Trp、Met-Arg、Pro-Phe、Val-Tyr、Val-Met)，以及10 mmol/L、20 mmol/L硫酸铵(pH 8.0)和pH 4.5+l-脯氨酸3种环境条件可显著促进立枯丝核菌菌核的形成；立枯丝核菌在pH 4.0-10.0范围内均能形成菌核；KEGG分析显示，诱导菌核形成的物质主要参与了代谢途径、ABC转运蛋白、次生代谢物生物合成和d-氨基酸代谢等通路。【结论】营养限制和环境胁迫是诱导立枯丝核菌菌核形成的关键因素。在营养受限的条件下，适宜立枯丝核菌形成菌核的物质包括d-山梨醇、d-木糖、N-乙酰-d-半乳糖胺、d-阿拉伯糖和d-松三糖等5种碳源；N-乙酰-d-氨基葡萄糖、腺苷和胸腺嘧啶等3种氮源；尿苷3′-单磷酸盐和磷酸胆碱等2种磷源；吐温-80等1种营养补充物；以及Arg-Trp、Met-Arg、Pro-Phe、Val-Tyr和Val-Met等5种肽类氮源。适宜的渗透压环境为10 mmol/L、20 mmol/L硫酸铵(pH 8.0)，适宜的pH环境为pH 4.0-4.5和pH 9.5-10.0。本研究结果为立枯丝核菌菌核形成机制的研究奠定了基础。

关键词

立枯丝核菌 / 菌核 / 物质代谢 / 表型芯片 / KEGG通路

Abstract

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[Objective] To analyze the metabolic substrates required for the sclerotial formation of Rhizoctonia solani and understand the influence of nutritional elements and environmental factors on this process. [Methods] Biolog phenotypic microarray was used to study the effects of 663 nutritional substances, 96 osmotic environments, and 96 pH environments on the sclerotial formation of R. solani. [Results] Among the tested nutritional substances and environmental conditions, 19/95 carbon sources, 21/95 nitrogen sources, 16/94 phosphorus and sulfur sources, 69/94 nutritional supplements, 61/282 peptide nitrogen sources, 28/96 osmotic environments, and 40/96 pH environments induced the sclerotial formation of R. solani. Notably, N-acetyl-d-glucosamine, uridine 3′-monophosphate, phosphoryl choline, and five dipeptides (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met), as well as three environmental conditions of 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and pH 4.5+l-proline, significantly induced the sclerotial formation of R. solani. R. solani formed sclerotia in the environments with a broad range of pH 4.0-10.0. The KEGG analysis indicated that the substances inducing sclerotial formation were primarily involved in metabolic pathways, ABC transporters, secondary metabolite biosynthesis, and d-amino acid metabolism. [Conclusion] Nutrient limitation and environmental stress are key factors inducing the sclerotial formation of R. solani. Under nutrient-restricted conditions, the suitable substances for inducing sclerotial formation include five carbon sources (d-sorbitol, d-xylose, N-acetyl-d-galactosamine, d-arabinose, and d-melezitose), three nitrogen sources (N-acetyl-d-glucosamine, adenosine, and thymidine), two phosphorus sources (uridine 3′-monophosphate and phosphoryl choline), one nutritional supplement (Tween-80), and five peptide nitrogen sources (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met). The suitable osmotic environments were 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and the suitable pH environments were pH 4.0-4.5 and pH 9.5-10.0. These findings provide a foundation for understanding the sclerotial formation mechanism of R. solani.

Key words

Rhizoctonia solani / sclerotium / material metabolism / phenotypic microarray / KEGG pathway

引用本文

向立刚, 汪汉成, 蔡刘体, 陈丽莉, 张文建, 王军, 孟建玉, 杨靓, 温明霞. 基于Biolog表型芯片的立枯丝核菌菌核形成所需代谢底物分析. 微生物学报, 2025 , 65 (5) : 2190 -2200 . DOI: 10.13343/j.cnki.wsxb.20240778

Ligang XIANG, Hancheng WANG, Liuti CAI, Lili CHEN, Wenjian ZHANG, Jun WANG, Jianyu MENG, Liang YANG, Mingxia WEN. Biolog phenotypic microarray reveals the metabolic substrates required for sclerotial formation of Rhizoctonia solani[J]. Acta Microbiologica Sinica, 2025 , 65 (5) : 2190 -2200 . DOI: 10.13343/j.cnki.wsxb.20240778

正文

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立枯丝核菌(Rhizoctonia solani Kühn)，隶属于担子菌门(Basidiomycota)，最早由Kühn于1858年在马铃薯上发现^[1]。目前，立枯丝核菌可分为14个融合群(anastomosis groups, AGs)^[2]，部分融合群又根据其形态、寄主范围以及DNA序列的差异被细分为不同亚群^[3-4]。作为一种典型的土传真菌，该菌能够侵染包括绝大多数谷类作物、蔬菜、水果(或果树)以及观赏植物在内的广泛寄主，引起种腐、根腐、冠腐、茎腐、纹枯和立枯等多种病症^[5]。立枯丝核菌因其广泛的寄主范围和多样的病害类型，已成为农业生产中影响最广的病原菌之一。

菌核是部分真菌在生长条件受限或受到不良环境胁迫时，由营养菌丝聚集形成的一种特殊休眠体结构，其形态多样，能在环境条件适宜时萌发，形成无性或有性繁殖结构^[6]。立枯丝核菌的菌核形状不规则，表面粗糙坚硬，多呈浅褐色至黑褐色，且内外颜色一致。由于菌核的存在，立枯丝核菌可以适应多种极端环境，并抵御杀菌剂的胁迫，从而增加菌株的抗药性^[7-8]。据报道菌核可在土壤或植物残体中存活多年，待环境条件适宜时又能迅速萌发，成为初侵染源侵染植物^[9-10]。菌核的存在无疑为农业生产中立枯丝核菌所引发的病害防治带来了巨大挑战。

虽然菌核能够抵御多种不良环境，但其形成也受温度、光照、pH、活性氧(reactive oxygen species, ROS)以及营养物质等环境因子的影响^[11-14]。Biolog表型芯片技术是一种与基因组学和蛋白组学并列的技术，能够同时测定微生物在190种碳源、95种氮源、282种肽类、94种磷硫源、94种营养补充物，以及96种渗透压和96种pH环境下的代谢情况。因此，本研究利用Biolog表型芯片技术分析立枯丝核菌菌核形成过程中所需的代谢底物，旨在了解营养元素和环境因子对其菌核形成的影响，并为通过抑制菌核形成来进行病害防控技术的研究提供参考依据。

1 材料与方法

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1.1 材料

供试立枯丝核菌LK4菌株分离自贵州省遵义市染病烟苗。d-葡萄糖、无水磷酸二氢钾和硫酸钠均购自西格玛奥德里奇(上海)贸易有限公司；酵母氮源购自BD医疗器械有限公司；0.2 μm注射器过滤膜购自颇尔生命科学有限公司；OmniLog PM高通量微生物细胞表型测定系统、浊度计、FF-IF接种液和表型微阵列(Phenotype MicroArray, PM)微孔板PM1、PM2A、PM3B、PM4A、PM5、PM6、PM7、PM8、PM9、PM10均购自Biolog公司。

1.2 试剂及菌丝悬浮液制备

d-葡萄糖溶液：准确称量57.664 g d-葡萄糖用灭菌水溶解并定容至100 mL。PM (3B、5、6、7、8)添加液：称取0.816 6 g无水磷酸二氢钾和0.341 g硫酸钠用灭菌水溶解定容至100 mL。PM (9、10)添加液：称取8.04 g酵母提取物用灭菌水溶解并定容至100 mL。将d-葡萄糖溶液和2种PM添加液分别使用0.2 μm过滤膜过滤除菌。立枯丝核菌菌丝片段的制备：将PDB中静置培养72 h的立枯丝核菌菌丝用无菌纱布过滤，去除菌丝包裹的菌碟后，用灭菌蒸馏水冲洗菌丝去除其携带的培养基，反复冲洗3遍后将菌丝置于灭菌研钵中，加入适量灭菌水充分研磨以制成菌丝片段悬浮液。最后使用浊度仪测量并调节悬浮液浊度至62%。

1.3 微孔板接种及孵育

各微孔板接种成分如表1所示，PM1和PM2A微孔板接种成分相同，PM3B、PM5、PM6、PM7和PM8微孔板接种成分相同，PM9和PM10微孔板接种成分相同，PM4A微孔板与其他微孔板不同。采用8通道移液器将配制好的混合接种液加入到相应微孔板中，每孔100 μL。微孔板完成接种并经封口膜封闭后，置于OmniLog PM系统中28 ℃孵育240 h。

1.4 菌核形成能力评估

培养结束后，综合评估各微孔中菌核的数量及体积大小，以评价菌核的形成情况。菌核形成能力等级划分如下：++++表示极强；+++表示强；++表示一般；+表示弱。

2 结果与分析

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2.1 碳源对立枯丝核菌菌核形成的影响

在95种供试碳源中共有19种可以诱导立枯丝核菌形成菌核，其中在d-山梨醇(d-sorbitol)、d-木糖(d-xylose)、N-乙酰-d-半乳糖胺(N-acetyl-d-galactosamine)、d-阿拉伯糖(d-arabinose)和d-松三糖(d-melezitose)诱导下菌核形成能力一般；而在其余14种碳源诱导下，菌核形成能力较弱(表2)。KEGG分析结果表明，诱导菌核形成的碳源主要参与的代谢通路包括代谢途径(metabolic pathways)、ABC转运蛋白(ABC transporters)、戊糖和葡萄糖醛酸相互转化(pentose and glucuronate interconversions)、半乳糖代谢(galactose metabolism)和次生代谢物生物合成(biosynthesis of secondary metabolites)等(原始数据存储在国家微生物科学数据中心，网址为http://nmdc.cn，编号为NMDCX0001772)。

2.2 氮源对立枯丝核菌菌核形成的影响

立枯丝核菌在PM3B微孔板中的产核情况表明，22%的供试氮源(21/95)可诱导菌核形成，其中N-乙酰-d-氨基葡萄糖(N-acetyl-d-glucosamine)诱导下的菌核形成能力最强，腺苷(adenosine)和胸腺嘧啶(thymidine)次之(表3)。KEGG分析结果表明，N-乙酰-d-氨基葡萄糖参与氨基糖和核苷酸糖代谢(amino sugar and nucleotide sugar metabolism)、代谢途径、核苷酸糖的生物合成(biosynthesis of nucleotide sugars)、ABC转运蛋白和磷酸转移酶系统(phosphotransferase system, PTS) 5条代谢通路。诱导菌核形成的氮源主要参与的代谢通路包括代谢途径、ABC转运蛋白、次生代谢物生物合成和d-氨基酸代谢(d-amino acid metabolism)等(编号为NMDCX0001772)。

2.3 磷、硫源对立枯丝核菌菌核形成的影响

磷、硫作为细胞结构的组成成分以及酶活性的参与者，在94种供试磷、硫源中仅有16种能够诱导立枯丝核菌产生菌核(表4)。其中，立枯丝核菌在尿苷3′-单磷酸盐(uridine 3′- monophosphate)和磷酸胆碱(phosphoryl choline)诱导下的菌核形成能力极强。KEGG分析显示，尿苷3′-单磷酸盐参与的代谢通路包括代谢途径和嘧啶代谢(pyrimidine metabolism)，而磷酸胆碱参与的代谢通路包括壁聚糖酸生物合成(teichoic acid biosynthesis)、甘油磷脂代谢(glycerophospholipid metabolism)、代谢途径和癌症中的胆碱代谢(choline metabolism in cancer) (编号为NMDCX0001772)。

2.4 营养补充物质对立枯丝核菌菌核形成的影响

在PM5微孔板中可诱导立枯丝核菌形成菌核的物质数量最多，占供试营养补充物的73% (69/94)，但其诱导菌核形成的能力大多为一般或较弱(表5)。KEGG分析显示，诱导菌核形成的营养补充物质主要参与的代谢通路包括代谢途径、ABC转运蛋白、次生代谢物生物合成、辅因子生物合成(biosynthesis of cofactors)、核苷酸代谢(nucleotide metabolism)和氨基酸生物合成(biosynthesis of amino acids)等(编号为NMDCX0001772)。

2.5 肽类氮源对立枯丝核菌菌核形成的影响

PM6、PM7和PM8微孔板用于检测肽类氮源对立枯丝核菌菌核形成的影响。结果表明，在282种供试肽类氮源中，共有61种可诱导立枯丝核菌形成菌核，其中Arg-Trp、Met-Arg、Pro-Phe、Val-Tyr和Val-Met等5种肽类氮源诱导菌核形成的能力极强；Ala-Leu、Ile-Phe、Ile-Tyr、Phe-Pro、Ala-Val和γ-Glu-Gly等6种肽类氮源诱导菌核形成的能力较强(表6)。

2.6 渗透压环境对立枯丝核菌菌核形成的影响

在96种渗透压环境中，共有28种可诱导立枯丝核菌产生菌核，其中10 mmol/L和20 mmol/L硫酸铵(ammonium sulfate)诱导菌核形成能力最强，其次是60 mmol/L硝酸钠(sodium nitrate) (表7)。KEGG分析表明，在28种渗透压环境中，仅磷酸钠(sodium phosphate)、苯甲酸钠(sodium benzoate)、乙二醇(ethylene glycol)和尿素(urea)参与了代谢通路，参与数量分别为10、10、4和11 (编号为NMDCX0001772)。

2.7 pH环境对立枯丝核菌菌核形成的影响

如表8所示，立枯丝核菌在pH 4.0-10.0的范围内均能产生菌核，且在pH 4.0、4.5、9.5和10.0时，菌核形成能力较强；在pH 5.0-9.0时，菌核形成能力一般或较弱。此外，在pH 4.5+l-脯氨酸(l-proline)环境下立枯丝核菌的菌核形成能力最强(图1)。在诱导菌核形成的pH环境中，共有18种底物参与了代谢通路，主要包括代谢途径、氨基酸生物合成、次生代谢物生物合成、蛋白质消化和吸收(protein digestion and absorption)、ABC转运蛋白和氨酰基tRNA生物合成(aminoacyl-tRNA biosynthesis)等(编号为NMDCX0001772)。

3 讨论

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营养限制是诱导菌核形成的重要因素^[15]。Christias等^[16]发现，在营养充足的条件下，核盘菌的菌丝体得以持续生长，但菌核形成时间显著推迟。Cooke等^[17]研究表明，在营养受限的条件下，菌株会优先将有限的营养物质集中用于菌核的形成，以增强其抵御不良环境的能力。在本研究中，微孔板各孔中预先填充了极少量的营养物质，接种100 μL接种液后，立枯丝核菌即处于营养受限状态。因此，能够在该条件下诱导菌核形成的物质，必定在菌核形成过程中发挥重要作用。N-乙酰-d-氨基葡萄糖、尿苷3′-单磷酸盐、磷酸胆碱和5种二肽(Arg-Trp、Met-Arg、Pro-Phe、Val-Tyr、Val-Met)已被证实在营养受限的微孔环境中显著诱导立枯丝核菌的菌核形成。此外，其他178种物质也可诱导立枯丝核菌菌核形成，但诱导能力存在差异。KEGG分析表明，这些物质主要参与了代谢途径、ABC转运蛋白、次生代谢物生物合成和d-氨基酸代谢等常规代谢通路。相比之下，显著诱导菌核形成的N-乙酰-d-氨基葡萄糖、尿苷3′-单磷酸盐和磷酸胆碱还参与了氨基糖和核苷酸糖代谢、核苷酸糖的生物合成、磷酸转移酶系统、嘧啶代谢、壁聚糖酸生物合成和甘油磷脂代谢等特有代谢通路。

ROS在菌核形成过程中起着关键作用。它们在真核生物的正常新陈代谢中产生，并在生物体内维持低浓度以避免对细胞造成损害^[18]。研究表明，菌丝向菌核分化时ROS产量增加，其积累可能诱导菌核形成，同时菌核形成消耗ROS以保护细胞免受其毒性影响^[19-20]。渗透压环境的改变往往会引起微生物的氧化应激反应，导致细胞内氧化还原失衡，ROS增加。本研究发现，PM9微孔板中28种渗透压环境可诱导菌核形成，其中硫酸铵、硝酸钠和乳酸钠诱导菌核的能力随浓度增加先强后弱。这可能是由于高浓度情况下产生的ROS超过了细胞消耗的阈值，导致细胞受损，最终影响菌核的形成。至于磷酸钠、氯化钾、硫酸钠和乙二醇诱导菌核形成能力不受其浓度影响的原因尚待进一步研究。

对于环境pH，普遍认为偏酸性环境有利于菌核形成，而中性或碱性环境会抑制其形成。然而，本研究发现立枯丝核菌在pH 4.0-10.0的范围内均能形成菌核，这可能是由于菌株通过代谢活动改变了培养环境的pH值。具体而言，在中等碱性环境(pH 8.0-9.0)中菌株可降低培养基的pH值，创造适宜的酸性环境以促进菌核形成^[12,21]。在PM10微孔板中，立枯丝核菌在pH 4.5且添加脯氨酸的环境中菌核形成能力最强，这可能归因于2个因素：(1) pH 4.5提供了适宜的酸性环境，有利于立枯丝核菌菌核形成；(2) 脯氨酸在微生物抵御不良环境中具有多重功能，包括作为渗透压调节物质维持细胞水分平衡、与蛋白质结合形成保护膜以保护生物大分子的结构与活性，以及减少ROS的积累，降低氧化应激对细胞的伤害。这些功能共同促进了立枯丝核菌的菌核形成。

4 结论

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本研究通过Biolog表型芯片技术，深入分析了立枯丝核菌菌核形成过程中对不同物质成分以及渗透压和pH环境的代谢响应。结果显示，在供试的物质及环境中分别有19/95的碳源、21/95的氮源、16/94的磷硫源、69/94的营养补充物、61/282的肽类氮源、28/96的渗透压环境以及40/96的pH环境(pH 4.0-10.0)可诱导立枯丝核菌形成菌核。适宜菌核形成的碳源包括d-山梨醇、d-木糖、N-乙酰-d-半乳糖胺、d-阿拉伯糖和d-松三糖；氮源包括N-乙酰-d-氨基葡萄糖、腺苷和胸腺嘧啶，磷源包括尿苷3′-单磷酸盐和磷酸胆碱；营养补充物质包括吐温-80；肽类氮源包括Arg-Trp、Met-Arg、Pro-Phe、Val-Tyr和Val-Met；适宜的渗透压环境为10 mmol/L和20 mmol/L硫酸铵(pH 8.0)；适宜的pH环境为pH 4.0-4.5和pH 9.5-10.0。可诱导立枯丝核菌形成菌核的物质主要参与的代谢通路包括代谢途径、ABC转运蛋白、次生代谢物生物合成和d-氨基酸代谢等。

基金

收起

国家自然科学基金(31960550)
国家自然科学基金(32460698)
国家自然科学基金(32070177)
贵州省科技项目(黔科合平台人才-CXTD[2023]021, ZK[2021]Key036, GCC[2022]028-1)
中国烟草总公司项目(110202101048(LS-08))
中国烟草总公司项目(110202001035(LS-04))
中国烟草总公司贵州省公司项目(2024XM06)

参考文献

收起

文献

收起

参考文献引证文献

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2025年第65卷第5期

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文章信息

doi: 10.13343/j.cnki.wsxb.20240778

接收时间：2024-12-03
首发时间：2026-02-05
出版时间：2025-05-04

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文章信息

作者

出版历史

收稿日期：2024-12-03
录用日期：2024-12-23

基金

National Natural Science Foundation of China(31960550)

国家自然科学基金(31960550)

National Natural Science Foundation of China(32460698)

国家自然科学基金(32460698)

National Natural Science Foundation of China(32070177)

国家自然科学基金(32070177)

Science and Technology Project of Guizhou Province (Qiankehe Talent Platform-CXTD[2023]021, ZK[2021]Key036, GCC[2022]028-1)

贵州省科技项目(黔科合平台人才-CXTD[2023]021, ZK[2021]Key036, GCC[2022]028-1)

China National Tobacco Corporation Project(110202101048(LS-08))

中国烟草总公司项目(110202101048(LS-08))

China National Tobacco Corporation Project(110202001035(LS-04))

中国烟草总公司项目(110202001035(LS-04))

China National Tobacco Corporation Guizhou Company Project(2024XM06)

中国烟草总公司贵州省公司项目(2024XM06)

作者信息

^1.贵州省烟草科学研究院，贵州贵阳

^2.贵州省烟草公司遵义市公司，贵州遵义

^3.福建农林大学，植物病毒研究所，福建福州

参考文献

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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20240778

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Solutions	PM1, 2A	PM3B, 5, 6,7, 8	PM4A	PM9, 10
FF-IF inoculum (mL)	20.00	50.00	10.00	20.00
d-glucose (mL)	-	1.88	0.38	0.75
PM additive solution (mL)	-	5.00	-	2.00
Mycelial fragment suspension (mL)	0.50	1.25	0.25	0.50
Sterile water (mL)	3.50	1.88	1.38	0.75
Total volume (mL)	24.00	60.00	12.00	24.00

Solutions

PM1, 2A

PM3B, 5, 6,7, 8

PM4A

PM9, 10

FF-IF inoculum (mL)

20.00

50.00

10.00

20.00

d-glucose (mL)

1.88

0.38

0.75

PM additive solution (mL)

5.00

2.00

Mycelial fragment suspension (mL)

0.50

1.25

0.25

0.50

Sterile water (mL)

3.50

1.88

1.38

0.75

Total volume (mL)

24.00

60.00

12.00

24.00

Microplate	Well	Substrate	Sclerotial formation
PM1	B02	d-sorbitol	++
B03	Glycerol	+
B08	d-xylose	++
C03	d,l-malic acid	+
C08	Acetic acid	+
D05	Tween-40	+
D10	Lactulose	+
E07	α-hydroxybutyric acid	+
F05	Fumaric acid	+
H06	l-lyxose	+
PM2A	B01	N-acetyl-d-galactosamine	++
B05	d-arabinose	++
C01	Gentiobiose	+
C04	d-melezitose	++
C12	Palatinose	+
D01	d-raffinose	+
D04	l-sorbose	+
D07	Turanose	+
G12	l-methionine	+

Microplate

Well

Substrate

Sclerotial formation

PM1

B02

d-sorbitol

B03

Glycerol

B08

d-xylose

C03

d,l-malic acid

C08

Acetic acid

D05

Tween-40

D10

Lactulose

E07

α-hydroxybutyric acid

F05

Fumaric acid

H06

l-lyxose

PM2A

B01

N-acetyl-d-galactosamine

B05

d-arabinose

C01

Gentiobiose

C04

d-melezitose

C12

Palatinose

D01

d-raffinose

D04

l-sorbose

D07

Turanose

G12

l-methionine

Well	Substrate	Sclerotial formation
A04	Nitrate	+
A10	l-aspartic acid	++
A12	l-glutamic acid	+
B01	l-glutamine	++
B10	l-serine	+
C01	l-tyrosine	+
C05	d-aspartic acid	+
C08	d-serine	+
D04	Hydroxylamine	+
D06	N-amylamine	+
D12	Agmatine	+
E08	d-glucosamine	+
E11	N-acetyl-d-glucosamine	++++
F03	Adenosine	+++
F04	Cytidine	+
F08	Thymine	+
F09	Thymidine	+++
H03	Ala-Glu	+
H04	Ala-Gly	+
H05	Ala-His	+
H10	Gly-Glu	++

Well

Substrate

Sclerotial formation

A04

Nitrate

A10

l-aspartic acid

A12

l-glutamic acid

B01

l-glutamine

B10

l-serine

C01

l-tyrosine

C05

d-aspartic acid

C08

d-serine

D04

Hydroxylamine

D06

N-amylamine

D12

Agmatine

E08

d-glucosamine

E11

N-acetyl-d-glucosamine

++++

F03

Adenosine

+++

F04

Cytidine

F08

Thymine

F09

Thymidine

+++

H03

Ala-Glu

H04

Ala-Gly

H05

Ala-His

H10

Gly-Glu

Well	Substrate	Sclerotial formation
A09	Adenosine 3′-monophosphate	+
B09	Guanosine 3′-monophosphate	+
C04	d-glucose-6-phosphate	++
D04	Phospho-l-arginine	+
D07	o-phospho-l-threonine	+
D09	Uridine 3′-monophosphate	++++
E04	Phosphoryl choline	++++
E11	Inositol hexaphosphate	+
F06	Dithiophosphate	+
G12	l-methionine sulfone	+
H02	Thiourea	+
H03	1-thio-β-d-glucose	+
H05	Taurocholic acid	+
H06	Taurine	+
H09	Butane sulfonic acid	+
H11	Methane sulfonic acid	+

Well

Substrate

Sclerotial formation

A09

Adenosine 3′-monophosphate

B09

Guanosine 3′-monophosphate

C04

d-glucose-6-phosphate

D04

Phospho-l-arginine

D07

o-phospho-l-threonine

D09

Uridine 3′-monophosphate

++++

E04

Phosphoryl choline

++++

E11

Inositol hexaphosphate

F06

Dithiophosphate

G12

l-methionine sulfone

H02

Thiourea

H03

1-thio-β-d-glucose

H05

Taurocholic acid

H06

Taurine

H09

Butane sulfonic acid

H11

Methane sulfonic acid

Well	Substrate	Sclerotial formation	Well	Substrate	Sclerotial formation
A06	l-aspartic acid	+	E07	β-alanine	+
A08	l-glutamic acid	+	E08	d-pantothenic acid	+
A10	Adenine	+	E09	Orotic acid	+
A11	Adenosine	++	E11	Uridine	+
A12	2′-deoxyadenosine	+	E12	2′-deoxyuridine	+
B01	l-glutamine	+	F01	Quinolinic acid	+
B02	Glycine	+	F02	Nicotinic acid	+
B04	l-isoleucine	+	F03	Nicotinamide	++
B06	l-lysine	+	F04	β-nicotinamide adenine dinucleotide	+
B08	l-phenylalanine	+	F05	δ-amino-levulinic acid	+
B09	Guanosine 3′,5′-cyclic monophosphate	+	F06	Hematin	+
B10	Guanine	+	F07	Deferoxamine	+
B11	Guanosine	+	F08	α-d-glucose	+
B12	2′-deoxyguanosine	+	F09	N-acetyl-d-glucosamine	+
C01	l-proline	+	F10	Thymine	+
C03	l-threonine	+	F11	Glutathione	+
C06	l-valine	+	F12	Thymidine	+
C07	l-isoleucine+l-valine	+	G01	Oxaloacetic acid	+
C08	Hydroxy-l-proline	++	G03	Cyanocobalamin	+
C09	(5)4-amino-imidazole-4(5)-carboxamide	+	G04	ρ-amino-benzoic acid	+
C10	Hypoxanthine	+	G05	Folic acid	+
C11	Inosine	+	G06	Inosine+thiamine	+
C12	2′-deoxyinosine	+	G08	Thiamine pyrophosphate	+
D02	l-citrulline	+	G10	Pyrrolo-quinoline quinone	+
D03	Chorismic acid	+	G12	Myo-inositol	++
D04	(-)Shikimic acid	+	H01	Butyric acid	+
D05	l-homoserine lactone	+	H03	α-ketobutyric acid	+
D07	d-aspartic acid	++	H04	Caprylic acid	+
D08	d-glutamic acid	+	H06	d,l-mevalonic acid	+
D10	Cytosine	+	H07	d,l-carnitine	+
D11	Cytidine	+	H08	Choline	++
D12	2′-deoxycytidine	+	H10	Tween-40	+
E01	Putrescine	+	H11	Tween-60	+
E02	Spermidine	+	H12	Tween-80	+++
E03	Spermine	+

Well

Substrate

Sclerotial formation

Well

Substrate

Sclerotial formation

A06

l-aspartic acid

E07

β-alanine

A08

l-glutamic acid

E08

d-pantothenic acid

A10

Adenine

E09

Orotic acid

A11

Adenosine

E11

Uridine

A12

2′-deoxyadenosine

E12

2′-deoxyuridine

B01

l-glutamine

F01

Quinolinic acid

B02

Glycine

F02

Nicotinic acid

B04

l-isoleucine

F03

Nicotinamide

B06

l-lysine

F04

β-nicotinamide adenine dinucleotide

B08

l-phenylalanine

F05

δ-amino-levulinic acid

B09

Guanosine 3′,5′-cyclic monophosphate

F06

Hematin

B10

Guanine

F07

Deferoxamine

B11

Guanosine

F08

α-d-glucose

B12

2′-deoxyguanosine

F09

N-acetyl-d-glucosamine

C01

l-proline

F10

Thymine

C03

l-threonine

F11

Glutathione

C06

l-valine

F12

Thymidine

C07

l-isoleucine+l-valine

G01

Oxaloacetic acid

C08

Hydroxy-l-proline

G03

Cyanocobalamin

C09

(5)4-amino-imidazole-4(5)-carboxamide

G04

ρ-amino-benzoic acid

C10

Hypoxanthine

G05

Folic acid

C11

Inosine

G06

Inosine+thiamine

C12

2′-deoxyinosine

G08

Thiamine pyrophosphate

D02

l-citrulline

G10

Pyrrolo-quinoline quinone

D03

Chorismic acid

G12

Myo-inositol

D04

(-)Shikimic acid

H01

Butyric acid

D05

l-homoserine lactone

H03

α-ketobutyric acid

D07

d-aspartic acid

H04

Caprylic acid

D08

d-glutamic acid

H06

d,l-mevalonic acid

D10

Cytosine

H07

d,l-carnitine

D11

Cytidine

H08

Choline

D12

2′-deoxycytidine

H10

Tween-40

E01

Putrescine

H11

Tween-60

E02

Spermidine

H12

Tween-80

+++

E03

Spermine

Microplate	Well	Substrate	Sclerotial formation	Microplate	Well	Substrate	Sclerotial formation
PM6	A05	Ala-Asn	+	PM7	D01	Phe-Pro	+++
A06	Ala-Glu	+	D10	Pro-Phe	++++
A09	Ala-Leu	+++	E04	Ser-Leu	+
A11	Ala-Phe	+	E06	Ser-Phe	+
B01	Ala-Ser	+	F04	Thr-Met	+
B02	Ala-Thr	+	F06	Trp-Ala	+
B05	Arg-Ala	+	G02	Trp-Ser	+
B07	Arg-Asp	++	H03	Val-Arg	++
B08	Arg-Gln	+	H04	Val-Asn	+
C02	Arg-Phe	++	H10	Val-Tyr	++++
C04	Arg-Trp	++++	PM8	A03	Ala-Asp	++
C07	Asn-Glu	+	A07	Ala-Val	+++
D05	Gln-Gln	+	A10	Asp-Gly	+
D06	Gln-Gly	++	A11	Glu-Ala	+
D07	Glu-Asp	+	A12	Gly-Asn	+
D08	Glu-Glu	+	B01	Gly-Asp	++
D09	Glu-Gly	+	B02	Gly-Ile	+
D10	Glu-Ser	+	C11	Pro-Arg	+
E01	Glu-Val	+	D07	Ser-Asn	+
E04	Gly-Cys	+	D08	Ser-Asp	+
E11	Gly-Pro	+	D10	Ser-Glu	+
F07	His-Leu	+	E02	Thr-Ser	++
G04	Ile-Arg	+	E08	Val-Glu	+
G05	Ile-Gln	+	E10	Val-Met	++++
G10	Ile-Phe	+++	F07	d-Ala-d-Ala	+
H02	Ile-Tyr	+++	G01	γ-Glu-Gly	+++
PM7	A02	l-glutamine	+	G03	Gly-d-Ala	++
B04	Lys-Trp	+	G04	Gly-d-Asp	+
B07	Met-Arg	++++	H04	Gly-Gly-Ile	+
C03	Met-Lys	+	H12	Tyr-Gly-Gly	+
C09	Phe-Ala	+

Microplate

Well

Substrate

Sclerotial formation

Microplate

Well

Substrate

Sclerotial formation

PM6

A05

Ala-Asn

PM7

D01

Phe-Pro

+++

A06

Ala-Glu

D10

Pro-Phe

++++

A09

Ala-Leu

+++

E04

Ser-Leu

A11

Ala-Phe

E06

Ser-Phe

B01

Ala-Ser

F04

Thr-Met

B02

Ala-Thr

F06

Trp-Ala

B05

Arg-Ala

G02

Trp-Ser

B07

Arg-Asp

H03

Val-Arg

B08

Arg-Gln

H04

Val-Asn

C02

Arg-Phe

H10

Val-Tyr

++++

C04

Arg-Trp

++++

PM8

A03

Ala-Asp

C07

Asn-Glu

A07

Ala-Val

+++

D05

Gln-Gln

A10

Asp-Gly

D06

Gln-Gly

A11

Glu-Ala

D07

Glu-Asp

A12

Gly-Asn

D08

Glu-Glu

B01

Gly-Asp

D09

Glu-Gly

B02

Gly-Ile

D10

Glu-Ser

C11

Pro-Arg

E01

Glu-Val

D07

Ser-Asn

E04

Gly-Cys

D08

Ser-Asp

E11

Gly-Pro

D10

Ser-Glu

F07

His-Leu

E02

Thr-Ser

G04

Ile-Arg

E08

Val-Glu

G05

Ile-Gln

E10

Val-Met

++++

G10

Ile-Phe

+++

F07

d-Ala-d-Ala

H02

Ile-Tyr

+++

G01

γ-Glu-Gly

+++

PM7

A02

l-glutamine

G03

Gly-d-Ala

B04

Lys-Trp

G04

Gly-d-Asp

B07

Met-Arg

++++

H04

Gly-Gly-Ile

C03

Met-Lys

H12

Tyr-Gly-Gly

C09

Phe-Ala

Well	Substrate	Sclerotialformation
D01	3% Potassium chloride	+
D02	4% Potassium chloride	+
D05	2% Sodium sulfate	+
D06	3% Sodium sulfate	++
D07	4% Sodium sulfate	+
D08	5% Sodium sulfate	++
D09	5% Ethylene glycol	+
D10	10% Ethylene glycol	++
D11	15% Ethylene glycol	+
D12	20% Ethylene glycol	++
E07	2% Urea	+
F01	1% Sodium lactate	++
F02	2% Sodium lactate	++
F11	11% Sodium lactate	+
G01	20 mmol/L Sodium phosphate pH 7.0	++
G02	50 mmol/L Sodium phosphate pH 7.0	+
G04	200 mmol/L Sodium phosphate pH 7.0	++
G05	20 mmol/L Sodium benzoate pH 5.2	++
G09	10 mmol/L Ammonium sulfate pH 8.0	++++
G10	20 mmol/L Ammonium sulfate pH 8.0	++++
G11	50 mmol/L Ammonium sulfate pH 8.0	++
G12	100 mmol/L Ammonium sulfate pH 8.0	++
H01	10 mmol/L Sodium nitrate	++
H02	20 mmol/L Sodium nitrate	+
H03	40 mmol/L Sodium nitrate	++
H04	60 mmol/L Sodium nitrate	+++
H05	80 mmol/L Sodium nitrate	++
H06	100 mmol/L Sodium nitrate	++

Well

Substrate

Sclerotialformation

D01

3% Potassium chloride

D02

4% Potassium chloride

D05

2% Sodium sulfate

D06

3% Sodium sulfate

D07

4% Sodium sulfate

D08

5% Sodium sulfate

D09

5% Ethylene glycol

D10

10% Ethylene glycol

D11

15% Ethylene glycol

D12

20% Ethylene glycol

E07

2% Urea

F01

1% Sodium lactate

F02

2% Sodium lactate

F11

11% Sodium lactate

G01

20 mmol/L Sodium phosphate pH 7.0

G02

50 mmol/L Sodium phosphate pH 7.0

G04

200 mmol/L Sodium phosphate pH 7.0

G05

20 mmol/L Sodium benzoate pH 5.2

G09

10 mmol/L Ammonium sulfate pH 8.0

++++

G10

20 mmol/L Ammonium sulfate pH 8.0

++++

G11

50 mmol/L Ammonium sulfate pH 8.0

G12

100 mmol/L Ammonium sulfate pH 8.0

H01

10 mmol/L Sodium nitrate

H02

20 mmol/L Sodium nitrate

H03

40 mmol/L Sodium nitrate

H04

60 mmol/L Sodium nitrate

+++

H05

80 mmol/L Sodium nitrate

H06

100 mmol/L Sodium nitrate

Well	Substrate	Sclerotial formation
A02	pH 4.0	+++
A03	pH 4.5	+++
A04	pH 5.0	++
A05	pH 5.5	++
A06	pH 6.0	+
A07	pH 7.0	+
A10	pH 9.0	++
A11	pH 9.5	+++
A12	pH 10.0	+++
B01	pH 4.5	+++
B02	pH 4.5+l-alanine	+
B03	pH 4.5+l-arginine	++
B04	pH 4.5+l-asparagine	++
B06	pH 4.5+l-glutamic acid	+
B07	pH 4.5+l-glutamine	+++
B08	pH 4.5+Glycine	+
B09	pH 4.5+l-histidine	+
C02	pH 4.5+l-phenylalanine	++
C03	pH 4.5+l-proline	++++
C04	pH 4.5+l-serine	++
C05	pH 4.5+l-threonine	+
C07	pH 4.5+l-citrulline	++
C08	pH 4.5+l-valine	+++
C09	pH 4.5+Hydroxy-l-proline	++
C10	pH 4.5+l-ornithine	++
C11	pH 4.5+l-homoarginine	++
C12	pH 4.5+l-homoserine	+
D02	pH 4.5+l-norleucine	++
D11	pH 4.5+Trimethylamine-N-oxide	+
E10	pH 9.5+l-isoleucine	++
F12	pH 9.5+l-homoserine	+
G03	pH 9.5+l-norvaline	++
H01	X-caprylate	+++
H05	X-β-d-galactoside	+
H06	X-α-d-glucuronide	+
H07	X-β-d-glucuronide	++
H08	X-β-d-glucosaminide	++
H09	X-β-d-galactosaminide	++
H10	X-α-d-mannoside	++
H12	X-SO₄	++

Well

Substrate

Sclerotial formation

A02

pH 4.0

+++

A03

pH 4.5

+++

A04

pH 5.0

A05

pH 5.5

A06

pH 6.0

A07

pH 7.0

A10

pH 9.0

A11

pH 9.5

+++

A12

pH 10.0

+++

B01

pH 4.5

+++

B02

pH 4.5+l-alanine

B03

pH 4.5+l-arginine

B04

pH 4.5+l-asparagine

B06

pH 4.5+l-glutamic acid

B07

pH 4.5+l-glutamine

+++

B08

pH 4.5+Glycine

B09

pH 4.5+l-histidine

C02

pH 4.5+l-phenylalanine

C03

pH 4.5+l-proline

++++

C04

pH 4.5+l-serine

C05

pH 4.5+l-threonine

C07

pH 4.5+l-citrulline

C08

pH 4.5+l-valine

+++

C09

pH 4.5+Hydroxy-l-proline

C10

pH 4.5+l-ornithine

C11

pH 4.5+l-homoarginine

C12

pH 4.5+l-homoserine

D02

pH 4.5+l-norleucine

D11

pH 4.5+Trimethylamine-N-oxide

E10

pH 9.5+l-isoleucine

F12

pH 9.5+l-homoserine

G03

pH 9.5+l-norvaline

H01

X-caprylate

+++

H05

X-β-d-galactoside

H06

X-α-d-glucuronide

H07

X-β-d-glucuronide

H08

X-β-d-glucosaminide

H09

X-β-d-galactosaminide

H10

X-α-d-mannoside

H12

X-SO₄