Pathogenicity of Fusarium spp. to chickpea seed and seedlings (Cicer arietinum L.)

Figures & data

Table 1.  Cotyledon and root disease severity rating scale used in evaluating disease symptoms of kabuli chickpea seeds and seedlings infected by Fusarium spp

Cotyledon rating scale	Description	Root rating scale	Description
1	Seed germinated, no disease apparent on cotyledons	1	Seed germinated, no disease apparent on roots
2	Seed germinated, < 25% of cotyledons necrotic or covered by mycelia	2	Seed germinated, < 25% of roots necrotic
3	Seed germinated, 25–50% of cotyledons necrotic or covered by mycelia	3	Seed germinated, 25–50% of roots necrotic
4	Seed germinated, 25–50% of cotyledons necrotic or covered by mycelia	4	Seed germinated, 50–75% of roots necrotic
5	Seed failed to germinate (due to disease) or 75–100% of cotyledons necrotic	5	Seed failed to germinate (due to disease) or 75–100% of roots necrotic

Table 2.  Pathogens isolated from kabuli chickpea affected by damping-off in Montana field trials. Pathogens were isolated from 12 diseased seeds at each location

Location	Fusarium only	Pythium only	Rhizopus only	F+P*	F+R*	P+R*	F+P+R*	Total Fusarium	Total Pythium	Total Rhizopus
Bozeman	2	1	0	4	1	1	3	10	9	5
Huntley	4	0	3	0	5	0	0	9	0	8
Sidney	1	1	0	6	2	0	2	11	9	4
Total	7	2	3	10	8	1	5	30	18	17
% Seed	19.4	5.6	8.3	27.8	22.2	2.8	13.9	83.3	50.0	47.2
Note: * Co-infections of Fusarium and Pythium (F+P); Fusarium and Rhizopus (F+R); Pythium and Rhizopus (P+R); and Fusarium, Pythium and Rhizopus (F+P+R).

Table 3.  Diversity of Fusarium isolates across three sites in Montana

Site	Total no. seeds tested	Richness*	Evenness^†	Dominant ITS species^‡
Bozeman	9	9	0.84	Giberella avenacea (67), F. tricinctum (67), F. oxysporum (33)
Huntley	10	8	0.82	Giberella avenacea (80), F. equiseti (50), F. tricinctum (30)
Sidney	8	9	0.83	F. oxysporum (63), F. solani (38), F. redolens (20)
Notes: * Number of unique ITS sequences per site.
† Simpson's index of diversity. Values close to 1 indicate the abundance of species is evenly distributed across the community.
‡ Per cent of seeds from which species were isolated is in parentheses.

Table 4.  Morphological and sequence-based identification of Fusarium spp. isolated from kabuli chickpea affected by damping-off in Montana

Isolatedesignation	1.3	3.5	3.2	4.6	DI	1.7	2.2	2.6
Location of origin	Bozeman	Bozeman	Huntley	Huntley	Huntley	Sidney	Sidney	Sidney
Morphological identification	F. solani	F. oxysporum	F. trincintum	F. culmorum	F. sambucinum	F. solani	F. solani	F. redolens
ITS sequence identification*
Accession no.	EU910075	EU910080	EU910079	EU910081	EU910082	EU910077	EU910076	EU910078
GenBank search return; accession number	Gibberella avenacea FA12; EU255802.1	Fusarium oxysporum isolate 25; EU839377.1	Fusarium acuminatum strain NRRL 54217; HM068325.1	Fusarium equiseti strain NRRL 13402; GQ505681.1	Fusarium equiseti strain NRRL 36478; GQ505743.1	Fusarium sp. SS-r3; GU797142.1	Fusarium oxysporum f. sp. phaseoli strain DM091019-1; HM756257.1	Fusarium redolens strain DH-A3; HM346540.1
Query coverage	100%	99%	100%	100%	100%	100%	99%	100%
E value	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
TEF sequence identification
^†Accession no.	EU910069	EU910072	FJ752547	EU910073	EU910074	EU910068	EU910070	EU910071
GenBank search return; accession number	Fusarium reticulatum var. negundinis isolate 15/2.3.1; DQ854864.1	Fusarium oxysporum f.sp. vasinfectum isolate IMI-338122; EU246573.1	Fusarium sp. K5225-2; AB294874.1	^§ Fusarium sp. DI; EU910074.1	^§ Fusarium sp. 4.6; EU910073.1	^‖ Fusarium sp. NRRL 44903; GU170620.1	^‖ Fusarium sp. NRRL 44903; GU170620.1	Fusarium redolens strain NRRL 52657; GU250584.1
Query coverage	95.42%	100%	100%	97%	97%	99%	99%	100%
E value	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
‡Fusarium-ID search return; isolate identification no.	Fusarium sp.; FC_01861_EF1a	Fusarium oxysporum species complex isolate NRLL 38608; MLST22	Fusarium tricinctum sp. complex isolate NRRL 36147; MLST2-a	No matching sequences	No matching sequences	Fusarium solani sp. complex isolate NRRL 32810; MLST5-m	Fusarium solani sp. complex isolate NRRL 32810; MLST5-m	Fusarium redolens; FD_01103_EF-1a
Query coverage	99.36%	99.83%	99.85%			100%	100%	99.845
Score	999.99	999.99	999.99			999.9	999.9	999.99
Notes: * ITS sequence amplified using primer pair ITS 1F and ITS 4. Blastn nr/nt query.
† Transcription elongation factor (TEF) 1-α sequence amplified using primer pair EF1 and EF2.blastn (nr/nt) query.
‡ TEF 1- α. Fusarium- ID query.
§ Closest identification other than isolates in this study had less than 25% coverage.
‖ Closest identification other than isolates in this study.

Fig. 1. Effects of moisture, inoculum density on disease incidence in chickpea seedlings across eight Fusarium spp. isolates under controlled conditions in growth chamber experiments. Inoculum densities are in propagules per gram of dry sand.

Table 5.  Analysis of variance and significance of contrasts for linear and quadratic trends of damping-off incidence of kabuli chickpea cause by Fusarium spp. in relation to moisture and inoculum density in growth chamber trials

	Source of variation			Soil moisture		Propagules per g soil*
Isolate	Soil moisture	Inoculum density	Soil moist. × inoc. density	Linear trend	Quadratic trend	Linear trend	Quadratic trend
1.3	< 0.0001	< 0.0001	< 0.0001	< 0.0001	0.0033	< 0.0001	< 0.0001
3.5	0.0005	< 0.0001	0.71	0.0166	0.1873	< 0.0001	0.9471
3.2	< 0.0001	< 0.0001	0.01	< 0.0001	0.0045	< 0.0001	< 0.0001
4.6	< 0.0001	< 0.0001	0.09	0.0005	0.2851	< 0.0001	0.0002
D.I.	0.17	< 0.0001	0.77	0.8474	0.2014	< 0.0001	0.0017
1.7	< 0.0001	< 0.0001	0.08	< 0.0001	0.7391	< 0.0001	< 0.0001
2.2	< 0.0001	< 0.0001	0.44	< 0.0001	0.1586	< 0.0001	0.0155
2.6	0.334	< 0.0001	0.10	0.8022	0.0881	< 0.0001	0.2013
Notes: For all isolates, the source of variation for soil moisture had 3 df, inoculum density had 4 df, and soil moisture × inoculum density had 12 df. All linear and quadratic trend contrasts have 1 df. Significant contrasts are in bold.
*Log propagules per g soil used in analysis.

Table 6.  Disease severity of cotyledons and roots infected by Fusarium spp. isolates and correlation between disease incidence and severity of damping-off of kabuli chickpea caused by Fusarium spp. in growth chamber trials

Isolate	Cotyledon*	Root*	Corr. incidence × disease severity (cotyledons) ^†	P-value	Corr. incidence × disease severity (roots) ^†	P-value
1.3	3.6 c	2.1  de	0.75	< 0.001	0.54	0.014
3.5	2.8 e	1.6 f	0.73	< 0.001	0.48	0.033
3.2	4.3 a	4.3 a	0.98	< 0.001	0.98	< 0.001
4.6	3.3 d	2.2 d	0.52	0.004	0.19	0.427
D.I.	4.0 b	2.6 c	0.73	< 0.001	0.50	0.009
1.7	3.2 d	3.0 b	0.65	0.002	0.88	< 0.001
2.2	2.3 f	2.4 cd	0.60	0.005	0.56	0.010
2.6	2.8 e	1.7 ef	0.45	0.047	0.31	0.180
Notes: * Numbers in a column for each isolate followed by the same letter are not significantly different at P < 0.05 (Fisher's LSD). Significant contrasts are in bold.
†Pearson's correlation coefficient (r).