Buckwheat seed quality and pathogenicity of <i>Fusarium</i> spp. in plants

Buckwheat is a species with great economic and production potential, which has gained increasing importance. This study aimed to determine the physical, physiological, and sanitary quality of samples from seed lots, and to evaluate the pathogenicity of Fusarium spp. in buckwheat plants. Physical and physiological quality was evaluated by the thousand-seed weight, moisture content, germination test (5^th and 7^th days), seedling length, and seedling dry mass, while sanitary quality was determined by the health test on filter paper, with the seeds subjected to asepsis and without asepsis. Isolates of Fusarium spp. were obtained from symptomatic seedlings in the paper roll germination test. To identify and characterize fungal isolates, morphological and molecular approaches were used. Pathogenicity was determined on healthy plants in a controlled environment. The lots showed high physiological quality in the germination evaluation (5^th and 7^th days). There was a high incidence of Fusarium spp. in all lots, which can be reduced with seed asepsis. The isolates were identified as Fusarium incarnatum-equiseti species complex, and were pathogenic to buckwheat plants.

Index terms:
damping-off; Fagopyrum esculentum Moench; germination; seed health

Authorship

Priscila Barbieri Zini ^**Corresponding author E-mail: priscilabarbieri88@hotmail.com

Departamento de Fitotecnia, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.Universidade Federal de Santa MariaBrazilSanta Maria, RS, BrazilDepartamento de Fitotecnia, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.

http://orcid.org/0000-0002-1530-7983

Tales Poletto

Departamento de Defesa Fitossanitária, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.Universidade Federal de Santa MariaBrazilSanta Maria, RS, BrazilDepartamento de Defesa Fitossanitária, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.

Vinícius Spolaor Fantinel

http://orcid.org/0000-0002-0414-7486

Nariane de Andrade

Departamento de Solos, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.Universidade Federal de Santa MariaBrazilSanta Maria, RS, BrazilDepartamento de Solos, Universidade Federal de Santa Maria, 97105-900, Santa Maria - RS, Brasil.

http://orcid.org/0000-0002-9277-6935

Ubirajara Russi Nunes

http://orcid.org/0000-0002-7124-9204

Marlove de Fátima Brião Muniz

http://orcid.org/0000-0001-7436-9589

Rodrigo Josemar Seminoti Jacques

http://orcid.org/0000-0002-8240-5145

*Corresponding author
E-mail: priscilabarbieri88@hotmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (8)

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Figure 1
Infected buckwheat seedlings observed in the paper roll germination test. (A) Infected seedlings in the paper roll test. (B) Observation through magnifying glass of the infected collar of the seedling.

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Figure 2
Morphological characteristics of Fusarium spp. associated with buckwheat seeds. (A - C) Culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 1, respectively; (D - F) culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 3, respectively; (G - I) culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 4, respectively; (J) culture in PDA medium of isolate 2; (L) monophialides; (M) polyphialides; (N) seedling damping-off in the pathogenicity test in sand; (O) seedlings after pathogenicity test (control on the left, inoculated on the right); (P) re-isolation after pathogenicity test.

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Figure 3
Phylogenetic tree obtained from the sequences of the partial gene elongation factor 1-alpha (tef1-α), of three isolates of Fusarium spp., associated with buckwheat (Fagopyrum esculentum Moench) seeds, showing the phylogenetic relationships of species in the Fusarium incarnatum-equiseti species complex (FIESC) according to the Maximum Likelihood statistical method. Numbers on the branches indicate the percentage of repetitions of bootstrap analysis in which the repetitions were observed (1000 repetitions).

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Table 1
Characterization of representative samples of buckwheat seed lots from different localities.

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Table 2
Moisture content (MC) and thousand-seed weight (TSW) of four samples of buckwheat seeds.

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Table 3
Means of germination (5^th day), germination (7^th day), abnormal seedlings and dead seeds of four buckwheat samples.

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Table 4
Means of root length (RL), shoot length (SL), root fresh mass (RFM), shoot fresh mass (SFM), root dry mass (RDM) and shoot dry mass (SDM) of four buckwheat samples.

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Table 5
Incidence of Fusarium sp. (FUS), Alternaria sp. (ALT), Cladosporium sp. (CLA), Colletotrichum sp. (COL), Aspergillus sp. (ASP) and Curvularia sp. (CUR) in buckwheat seeds subjected or not to asepsis.

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Table 6
Means of germination (5^th day), abnormal seedlings and dead seeds of four seed lots subjected or not to asepsis.

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Table 7
Means of germination (7^th day) of four buckwheat seed samples subjected to the methods with and without asepsis.

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Table 8
Morphological characteristics of Fusarium spp. isolated from parts of buckwheat seedlings after seven days of incubation in PDA (potato-dextrose-agar) medium, with 24-hour photoperiod, at 25 °C.

Figure 1 Infected buckwheat seedlings observed in the paper roll germination test. (A) Infected seedlings in the paper roll test. (B) Observation through magnifying glass of the infected collar of the seedling.

Figure 2 Morphological characteristics of Fusarium spp. associated with buckwheat seeds. (A - C) Culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 1, respectively; (D - F) culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 3, respectively; (G - I) culture in PDA medium; sporodochium in CLA medium and macroconidia of isolate 4, respectively; (J) culture in PDA medium of isolate 2; (L) monophialides; (M) polyphialides; (N) seedling damping-off in the pathogenicity test in sand; (O) seedlings after pathogenicity test (control on the left, inoculated on the right); (P) re-isolation after pathogenicity test.

Figure 3 Phylogenetic tree obtained from the sequences of the partial gene elongation factor 1-alpha (tef1-α), of three isolates of Fusarium spp., associated with buckwheat (Fagopyrum esculentum Moench) seeds, showing the phylogenetic relationships of species in the Fusarium incarnatum-equiseti species complex (FIESC) according to the Maximum Likelihood statistical method. Numbers on the branches indicate the percentage of repetitions of bootstrap analysis in which the repetitions were observed (1000 repetitions).

Table 1 Characterization of representative samples of buckwheat seed lots from different localities.

Sample	Cultivar	Origin	Harvest year
1	IPR 91- Baili	IAPAR^®^* * IAPAR: Agronomic Institute of Paraná. - PR	2018
2	IPR 92- Altar	IAPAR^®- PR	2018
3	IPR 91- Baili	Sementes com Vigor^® - RS	2018
4	IPR 92- Altar	Sementes Pozza^® - RS	2018

^*
IAPAR: Agronomic Institute of Paraná.

Table 2 Moisture content (MC) and thousand-seed weight (TSW) of four samples of buckwheat seeds.

Sample	MC (%)^* * Data not subjected to statistical analysis.	TSW (g)*
1	13.35	16.75
2	13	13.09
3	12.99	13.17
4	12	13.36

^*
Data not subjected to statistical analysis.

Table 3 Means of germination (5^th day), germination (7^th day), abnormal seedlings and dead seeds of four buckwheat samples.

	Germination (5^th day)	Germination (7^th day)	Abnormal	Dead
	.................................................................(%)...............................................................
Sample 1	96 A	99 A	1 A	0 D
Sample 2	71 B	89 B	7 A	4 B
Sample 3	71 B	82 B	8 A	10 A
Sample 4	76 B	95 A	4 A	1 C
CV (%)	7.89	8.58	64.86	33.0

Uppercase letters differ statistically in the column by the Scott-Knott test at 5% probability level. CV (%): coefficient of variation.

Table 4 Means of root length (RL), shoot length (SL), root fresh mass (RFM), shoot fresh mass (SFM), root dry mass (RDM) and shoot dry mass (SDM) of four buckwheat samples.

	RL	SL	RFM	SFM	RDM	SDM
	..............cm.............			................................g/10 pl^-1..............................
Sample 1	9.24 B	2.78 B	0.16 A	0.3 B	0.012 A	0.031 A
Sample 2	10.4 A	3.36 A	0.17 A	0.38 A	0.013 A	0.038 A
Sample 3	9.69 B	2.37 B	0.18 A	0.27 B	0.017 A	0.034 A
Sample 4	9.84 B	2.57 B	0.19 A	0.28 B	0.014 A	0.036 A
CV (%)	4.43	7.75	10.02	10.86	26.97	22.82

Uppercase letters differ statistically in the column by the Scott-Knott test at 5% probability level. CV (%): coefficient of variation.

Table 5 Incidence of Fusarium sp. (FUS), Alternaria sp. (ALT), Cladosporium sp. (CLA), Colletotrichum sp. (COL), Aspergillus sp. (ASP) and Curvularia sp. (CUR) in buckwheat seeds subjected or not to asepsis.

	Incidence (%)
	Without asepsis				With asepsis
	Samp. 1	Samp. 2	Samp. 3	Samp. 4	Samp. 1	Samp. 2	Samp. 3	Samp. 4
FUS	89 Aa	92 Aa	80 Aa	82 Aa	65 Ab	37 Bb	37 Bb	15 Cb
ALT	55 Aa	26 Ba	46 Aa	12 Ba	7 Ab	1 Ab	6 Ab	2 Ab
CLA	5 Ab	3 Aa	0 Aa	7 Aa	23 Aa	0 Ba	9 Aa	5 Aa
COL	5 Ba	29 Aa	0 Ba	3 Ba	0 Ba	12 Ab	0 Ba	0 Ba
ASP	3 Ba	0 Bb	0 Ba	41 Aa	3 Ba	20 Aa	3 Ba	2 Bb
CUR	0 Aa	0 Ab	0 Aa	0 Aa	1 Ba	7 Aa	0 Ba	2 Aa

Uppercase letters differentiate lot within each asepsis method and lowercase letters differentiate asepsis method within each lot by the Scott-Knott test at 5% probability level.

Table 6 Means of germination (5^th day), abnormal seedlings and dead seeds of four seed lots subjected or not to asepsis.

	Without asepsis				With asepsis
	(%)
	Samp. 1	Samp. 2	Samp. 3	Samp. 4	Samp. 1	Samp. 2	Samp. 3	Samp. 4
Germ. (5^th day)	91 Aa	83 Ba	64 Ca	82 Ba	57 Ab	64 Ab	38 Bb	17 Cb
Abnormal	2 Bb	5 Aa	10 Aa	6 Ab	6 Ba	7 Ba	19 Aa	25 Aa
Dead	2 Ba	6 Ba	13 Ab	3 Bb	2 Ca	11 Ba	24 Aa	7 Ba

Uppercase letters differentiate lot within each asepsis method and lowercase letters differentiate asepsis method within each lot by the Scott-Knott test at 5% probability level.

Table 7 Means of germination (7^th day) of four buckwheat seed samples subjected to the methods with and without asepsis.

	Without asepsis	With asepsis	Mean
	Germination (%)		Mean
Sample 1	97	93	95 a
Sample 2	89	82	86 b
Sample 3	74	56	65 c
Sample 4	91	68	80 b
Mean	88 a	75 b

Lowercase letters differ statistically in column and row by the Scott-Knott test at 5% probability level.

Table 8 Morphological characteristics of Fusarium spp. isolated from parts of buckwheat seedlings after seven days of incubation in PDA (potato-dextrose-agar) medium, with 24-hour photoperiod, at 25 °C.

Isolate	Diameter (mm)	^ADG* * ADG = Average daily growth; AGR = Average growth rate. (mm.day^-1)	AGR^* * ADG = Average daily growth; AGR = Average growth rate.	Sporulation (x10⁵ml^-1)	Conidium			Colony color
					Length	Width	Septum
					.............mm............
1	54.07	7.72	75.96	8.9	42.8	3.6	4 - 5	Light pink (5YR 8/2)
					(33.6 - 51.7)	(3 - 4.5)
2^ Isolate showed no sporulation.	37.9	5.41	49.93	-	-	-	-	Dark red (2.5 YR 3/6)
3	74.54	7.02	74.54	4.31	(34.6 - 56.9)	(2.9 - 4.5)	4 - 5	Pink (5YR 7/3)
4	17.8	2.54	16.44	4.76	30.9	3.1	3 - 4	Light red (2.5 YR 7/8)
					(24.6 - 39.9)	(2.5 - 4.1)

^*
ADG = Average daily growth; AGR = Average growth rate.
** Isolate showed no sporulation.

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Buckwheat seed quality and pathogenicity of <i>Fusarium</i> spp. in plants

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[1] *Corresponding author
E-mail: priscilabarbieri88@hotmail.com