Screening of historical and current Upland and Pima cotton germplasm for resistance to Rhizoctonia solani

Abstract

Objective

The fungus Rhizoctonia solani is an important seedling pathogen to many plant hosts including cotton (Gossypium). For multiple annual crops there have been relatively few screenings of germplasm conducted to identify potential sources of resistance to R. solani, and for cotton we have not been able to identify any recent germplasm screenings for resistance to this seedling pathogen. Therefore, the objective of this study was to screen historical as well as more recently developed Upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) cotton germplasm for resistance/susceptibility to R. solani.

Results

The results of the R. solani screening found no significant differences among 85 Upland and 10 Pima cotton genotypes, which were all similarly susceptible to R. solani based on data for root rot and fresh root weight. While Upland and Pima cotton make up the majority of cotton grown worldwide, the lack of resistance identified in both historical and newer Upland and Pima germplasm suggests a pressing need for further exploration and selection of novel sources of resistance within the vast genetic diversity of different domesticated and wild cotton species.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13104-025-07161-y.

Introduction

Seedling pathogens cause some of the most limiting diseases that impact cotton (Gossypium spp.) production through all cotton growing regions [17]. The fungus Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris (A.B. Frank) is a ubiquitous soilborne fungus and a prominent seedling pathogen associated with the cotton seedling disease complex [11]. Despite substantial efforts in developing management strategies over the years, seedling diseases remain a significant challenge in cotton production [10].

Host plant resistance is a management tool that can be used to reduce the use of agrochemicals and can also be used in combination with other management strategies. However, to date, screening efforts have not identified high levels of resistance to R. solani or other seedling pathogens in cotton varieties cultivated in the United States [3, 5, 9, 13]. Most of these screening efforts were completed three or more decades ago and the current cotton varieties that are widely grown on a commercial scale have completely changed during that interval of time. Improved resistance to seedling disease in cotton varieties would improve cotton production in the U.S. Therefore, the objective of this study was to screen both historical and newer experimental (Pima and Upland) cotton germplasm for resistance/susceptibility to R. solani.

Main text

Materials and methods

Plant material

The plant material used for the phenotypic assay included 85 Upland and 10 Pima cotton genotypes that were obtained from the USDA-ARS Plant Stress and Germplasm Development Research Unit (PSGDRU), Lubbock, TX. Seed for the new germplasm/breeding lines were increased in the greenhouse or field in California as described by Ulloa et al. [14] and SA lines were previously provided to PSGDRU by the USDA-ARS Cotton Germplasm Collection, College Station, TX, and seed was increased in the greenhouse and under field conditions at PSGDRU. During seed preparation (ginning and acid delinting) for planting, seed lots for most of the germplasm lines were not hot water treated. However, a few lines were treated to check for difference in germination or emergence. Hot water treatment is usually applied to seed after acid delinting to break seed dormancy or rapid sprouting/increase germination wild and photoperiod-sensitive cotton. The treatment consists of placing seed lots in a water bath at around 100^o C for 1 to 2 min before seed drying. For this specific study, no significant difference was found between germplasm lines. Some lines denotated ‘*’ in Table 1 are duplicates that have seed with hot water treatment (HWT). The germplasm represented lines that were released between the 1930’s to 2020 from both private and public breeding programs (Table 1). The new Upland and Pima plant material were newly developed progenies that were used for breeding for Fusarium wilt (Fusarium oxysporum f. sp vasinfectum) race 4 resistance [15, 16].

Table 1.

Cotton germplasm used in this study to evaluate their response when inoculated with Rhizoctonia solani

Germplasm ID	Pedigree 1	PI No	Pedigree 2	Cotton	Gossypium
13IN8218	SA-1078	529,147	ACALA, N.M. 8893	Upland	G. hirsutum
13IN8219	SA-0464	528,772	AHA 6-1-4 (ACALA x HOPI x ACALA)	Upland	G. hirsutum
12TXU0033-36	SA-1626	606,810	Acala_SJ-2	Upland	G. hirsutum
WS2018	SA-2289	590,568	Acala_NemX	Upland	G. hirsutum
13IN8227	SA-1148	529,214	AUBURN M	Upland	G. hirsutum
13IN8231	SA-1066	529,135	BN	Upland	G. hirsutum
13IN8236	SA-1150	529,216	COKER 100 A (WR)	Upland	G. hirsutum
13IN8238	SA-0450	528,760	COKER 100 STAPLE	Upland	G. hirsutum
13IN8239	SA-1155	529,221	DEKALB 108	Upland	G. hirsutum
13IN8241	SA-1154	529,220	DELA QUEEN	Upland	G. hirsutum
13IN8242	SA-0453	528,763	DELFOS 3506	Upland	G. hirsutum
13IN8243	SA-0452	528,762	DELFOS 4	Upland	G. hirsutum
13IN8247	SA-1151	529,217	DELFOS 9169	Upland	G. hirsutum
13IN8249	SA-0454	528,764	DELFOS 651	Upland	G. hirsutum
13IN8250	SA-1058	529,127	DELFOS 8274	Upland	G. hirsutum
13IN8246	SA-0459	528,768	DELTAPINE 12	Upland	G. hirsutum
13IN8251	SA-0460	528,769	DELTAPINE 14(44–51)	Upland	G. hirsutum
13IN8297	SA-1121	529,187	M8 (DOUBLE HAPLOID DELTAPINE 14)	Upland	G. hirsutum
13IN8252	SA-0462	528,770	DELTAPINE 15	Upland	G. hirsutum
13IN8254	SA-0458	528,767	DELTAPINE A	Upland	G. hirsutum
13IN8255	SA-1153	529,219	DELTAPINE SMOOTH LEAF	Upland	G. hirsutum
13IN8258	SA-1465	529,519	DES 422	Upland	G. hirsutum
13IN8262	SA-1220	529,285	DES 21326-04	Upland	G. hirsutum
13IN8263	SA-1600	518,762	DES 422 (Sub Okra)	Upland	G. hirsutum
13IN8263 HWT*	SA-1600 HWT*	518,762	DES 422 (Sub Okra)	Upland	G. hirsutum
13IN8264	SA-1466	529,520	DES 56	Upland	G. hirsutum
13IN8266	SA-0998	529,067	DES 716	Upland	G. hirsutum
13IN8272	SA-1645	536,524	DES 936	Upland	G. hirsutum
13IN8273	SA-1218	529,283	DES 89-11-10	Upland	G. hirsutum
13IN8274	SA-1643	536,522	DES 920	Upland	G. hirsutum
13IN8275	SA-0742	528,933	DWARF I FREGO (seg. fg)	Upland	G. hirsutum
13IN8276	SA-0467	528,774	EMPIRE P 45 − 10	Upland	G. hirsutum
13IN8284	SA-0743	528,934	FREGO UPLAND CR. DW. MEADE (no fg)	Upland	G. hirsutum
13IN8285	SA-1095	529,163	FOX BIG BOLL	Upland	G. hirsutum
13IN8285 HWT*	SA-1095 HWT*	529,163	FOX BIG BOLL	Upland	G. hirsutum
13IN8287	SA-1094	529,163	H.A. 11	Upland	G. hirsutum
13IN8291	SA-0677	528,888	LINTSING STONEVILLE	Upland	G. hirsutum
13IN8294	SA-1090	529,159	MS-1	Upland	G. hirsutum
13IN8295	SA-1599	518,768	MD 65 − 11 (Sub Okra)	Upland	G. hirsutum
PS-590 12TXU038	SA-2193	566,941	MD51ne Okra	Upland	G. hirsutum
PS-591 12TXU044	FM832		Fiber Max 832 Okra	Upland	G. hirsutum
13IN8302	SA-1160	529,226	REX SL	Upland	G. hirsutum
13IN8303	SA-1598	606,807	RRB2-10 (Sub Okra)	Upland	G. hirsutum
13IN8312	SA-0437	528,751	STONEVILLE 2	Upland	G. hirsutum
13IN8314	SA-1163	529,229	STONEVILLE 213	Upland	G. hirsutum
13IN8319	SA-0308	528,654	STONEVILLE 2B (ORIGINAL)	Upland	G. hirsutum
13IN8320	SA-0476	528,782	STONEVILLE 2B-7	Upland	G. hirsutum
13IN8321	SA-1334	529,390	STONEVILLE 256 (79256)	Upland	G. hirsutum
13IN8336	SA-1031	529,100	STONEVILLE 508	Upland	G. hirsutum
13IN8338	SA-0312	528,658	STONEVILLE 5 A	Upland	G. hirsutum
13IN8339	SA-0549	528,838	STONEVILLE 5 A T 219-6	Upland	G. hirsutum
13IN8342 HWT*	SA-1221 HWT*	529,286	STONEVILLE 618 BBR	Upland	G. hirsutum
13IN8342	SA-1221	529,286	STONEVILLE 618 BBR	Upland	G. hirsutum
13IN8344	SA-1190	529,255	STONEVILLE 603	Upland	G. hirsutum
13IN8345	SA-0542	528,835	STONEVILLE 5 A, T 226 − 11	Upland	G. hirsutum
13IN8347	SA-0472	528,778	STONEVILLE 62	Upland	G. hirsutum
13IN8350	SA-1333	529,389	STONEVILLE 731 N (98731)	Upland	G. hirsutum
13IN8356	SA-0048	528,461	STONEVILLE CLEAN SEED	Upland	G. hirsutum
13IN8357	SA-1470	529,524	STONEVILLE 825	Upland	G. hirsutum
13IN8359	SA-0380	528,706	STONEVILLE X HOPI 12-1-2-1	Upland	G. hirsutum
STV474	Stoneville 474		Stoneville 474	Upland	G. hirsutum
13IN8368	SA-0473	528,779	WASHINGTON	Upland	G. hirsutum
14TUN119.BULK	MCU5		MCU5	Upland	G. hirsutum
14TUN219.BULK	MCU5		MCU5	Upland	G. hirsutum
NM67	NM67		NM67	Upland	G. hirsutum
PHY72	Phytogen 72		Phytogen 72	Upland	G. hirsutum
TM1	Upland TM-1		Upland TM-1	Upland	G. hirsutum
FBCX-2	PS-588 12TXU022		FBCX-2	Upland	G. hirsutum
Shorty	PS-589 12TXU026		Shorty	Upland	G. hirsutum
Coker 312	Upland Coker 312		Coker 312	Upland	G. hirsutum
PSS-U77B	Upland PSS-U77B		SA3208 X PS-592 (PimaS6 x PimaUzbek)	Upland	G. hirsutum
Sel-1002	62_MU2019		F1xF4 (SA-1643 x NM12Y1004) x (F4-Shorty x FBCX-2)	Upland	G. hirsutum
Sel-1006	66_MU2019		F1xF4 (SA-1643 x NM12Y1004) x (F4-Shorty x FBCX-2)	Upland	G. hirsutum
Sel-1007	74_MU2019		F1xF4 (SA-1643 x NM12Y1004) x (F4-Shorty x FBCX-2)	Upland	G. hirsutum
Sel-1009	115_MU2019		F1xF4 (SA-1643 x NM12Y1004) x (F4-Shorty x FBCX-2)	Upland	G. hirsutum
Sel-1023	88_MU2019		SA-0072 (Mars Rose Cluster) x NM12Y1002	Upland	G. hirsutum
Sel-1024	5_MU2019		SA-1148 (Auburn M) x NM12Y1004	Upland	G. hirsutum
Sel-1025	8_MU2019		SA-1148 (Auburn M) x NM12Y1004	Upland	G. hirsutum
Sel-1029	9_MU2019		SA-1643 (DES 920) x NM12Y1004	Upland	G. hirsutum
Sel-1030	12_MU2019		SA-1643 (DES 920) x NM12Y1004	Upland	G. hirsutum
Sel-1035	17_MU2019		SA-1643 (DES 920) x NM12Y1004	Upland	G. hirsutum
Sel-1040	77_MU2019		SA3208 X PS-592 (PimaS6 x PimaUzbek)	Upland	G. hirsutum
Sel-1042	80_MU2019		SA3208 X PS-592 (PimaS6 x PimaUzbek)	Upland	G. hirsutum
Sel-1043	81_MU2019		SA3208 X PS-592 (PimaS6 x PimaUzbek)	Upland	G. hirsutum
Sel-1046	86_MU2019		SA3208 X PS-592 (PimaS6 x PimaUzbek)	Upland	G. hirsutum
Sel-1049	48_MU2019		SA3208XNM12Y1005	Upland	G. hirsutum
Sel-1050	49_MU2019		SA3208XNM12Y1005	Upland	G. hirsutum
Sel-1052	53_MU2019		SA3208XNM12Y1005	Upland	G. hirsutum
Sel-1015	2_MU2019		PS-592 (PimaS6 x PimaUzbek)	Pima	G. barbadense
Sel-1017	107_MU2019		PS592XSA3208	Pima	G. barbadense
Sel-1018	108_MU2019		PS592XSA3208	Pima	G. barbadense
SJ-10P20	09MU1032-33		Pima_SJ10	Pima	G. barbadense
SJ-10P14	09MU1314-15		SJ-FR05	Pima	G. barbadense
DP744	DeltaPine 744		DeltaPine 744	Pima	G. barbadense
DP340	DeltaPine 340		DeltaPine 340	Pima	G. barbadense
P-3-79	Pima 3–79		Pima 3–79	Pima	G. barbadense
PS7	Pima S7		Pima S7	Pima	G. barbadense
PS6	Pima S6		Pima S6	Pima	G. barbadense

Note- Germplasm with an * are duplicates of genotypes that have seed with and without hot water seed treatments

Inoculum preparation

The R. solani isolate RS1 used in the experiment was collected from symptomatic cotton in the Corcoran area of the San Joaquin Valley of California during the summer of 2017. Inoculum was prepared using oat seed infested with RS1 using the method described by Diaz et al. [1]. One modification was made where instead of inoculating the sterile oat seed with a conidial suspension, the sterile oat seed was inoculated with five colonized plugs (5-mm in diameter) from the edge of a R. solani colony grown on full strength potato dextrose agar (PDA) for 3 to 4 days at 25 °C. After three weeks, the colonized oats were air dried in a laminar flow hood and stored in paper bags at room temperature until used.

Phenotypic assay

The cotton germplasm was evaluated for resistance or susceptibility to R. solani in a greenhouse at the University of California’s Kearney Agricultural Research and Extension Center in Parlier, CA. The experimental design was a randomized complete block design with replications over time as the blocking factor due to the size of the experiment. There were a total of five replications (with the following start dates: October 25, 2021, March 4, 2022, April 22, 2022, June 2022, and August 1, 2022). Within a replication there was one inoculated (R. solani-infested-oat seed) pot and control (sterile non-inoculated oat seed) pot per cotton genotype, with each planted with eight cotton seeds. The non-inoculated control pots were not used in the final analysis but were used as a check to verify seed quality and germination for each of the cotton genotypes that were evaluated.

Briefly, pots (10.2 × 10.2 × 8.9 cm), were filled about two-thirds of the way to the top with potting mixture that consisted of 1 part peat moss (Sun Gro^® Horticulture, Seba Beach, Canada) and 1 part vermiculite (Thermo-O-Rock West Inc., Chandler, AZ). Next, a second layer of the R. solani-infested-oat seed or non-inoculated sterile oat seeds was measured using a sterile glass beaker (approximately 15 mL) and spread across the potting mixture. A third layer of the potting mixture (approximately 10 mL) was applied over the inoculum layer to provide a buffer zone between the seed and the inoculum to allow the seed to germinate before the roots interacted with the fungal inoculum. Lastly, a fourth layer of potting mixture (approximately 20 mL) was used to cover the seeds. The pots were then randomly arranged in the greenhouse. Temperatures in the greenhouse were maintained within the daily range of 18–24 °C and plants were watered as needed to avoid any water stress. At five weeks, data for stand counts, fresh shoot, and root weight, and a R. solani root rot rating were collected. Rhizoctonia solani symptoms were rated using a qualitative scale of 1 to 5 (1 = no root rot and 5 = preemergence damping-off with few if any roots) [2].

Statistical analysis

For the final analysis, R. solani root rot rating data was analyzed using the PROC MIXED of SAS version 9.4 (SAS Institute Inc., Cary, NC). Stand count data was converted by taking the total number of plants that germinated and dividing by the total number of seeds planted. Stands with a value of zero were converted to 0.01 and stands with a value of 1 were converted to 0.99. These values were then analyzed using a generalized linear mixed model (GLMM) with beta distribution in the PROC GLIMMIX procedure of SAS. For the fresh root weight data any weight of zero was converted to 0.01 and the data was analyzed using a lognormal response distribution in the PROC GLIMMIX procedure of SAS. The raw data that was analyzed is provided in the Additional File 1.

Results and discussion

This study evaluated a total of 85 Upland and 10 Pima cotton genotypes for their response to infection with R. solani. The germplasm that was evaluated was chosen to represent the genetic diversity found within cotton cultivars that have been grown in the United States from the 1930 until present (Table 1). To evaluate the germplasm, an oat seed inoculum was used as a carrier for the pathogen. This inoculation method was successful in fungal colonization and disease development in all 95 cotton genotypes evaluated. Of the 95 cotton genotypes that were evaluated very little to no range in response was observed among the cotton genotypes following inoculation with R. solani based on R. solani root rot rating, fresh root weight, and stand count (percent of germinated seedlings) data. The data means and standard errors for R. solani root rot rating, fresh root weight, and stand count for each cotton genotype are provided in Table 2. For the Rhizoctonia-inoculated cotton, based on the type three test of fixed effects no significant differences were observed among the cotton genotypes for both the root rot rating (F = 1.01, P = 0.4707) and fresh root weight (F = 1.06; P = 0.3378) data. It can be noted in Table 2 that in both Rhizoctonia-inoculated (F = 1.76, P < 0.0001) and non-inoculated control plants (F = 5.23, P < 0.0001) there were significant genotype differences in stand counts. However, across essentially all of the tested cotton genotypes, stand counts were significantly reduced when inoculated with R. solani in this study, with the highest average of 2.8 (35% germination) being observed in the Upland cotton Sel-1007 (Pedigree 1:74_MU2019, Pedigree 2:F1xF4 (SA-1643 x NM12Y1004) x (F4-Shorty x FBCX-2) (Table 2).

Table 2.

Stand counts, root rot, and fresh root weight averages and standard errors for Rhizoctonia solani inoculated and non-inoculated cotton germplasm

Germplasm ID	Cotton	Averages ± Standard Errors
		Inoculated			Non-inoculated
		Stand Count	Root Rot	Root Weight	Stand Count	Root Weight
13IN8218	Upland	1.0 ± 0.8	4.0 ± 0.6	0.5 ± 0.3	5.2 ± 0.9	1.4 ± 0.4
13IN8219	Upland	0.6 ± 0.4	3.8 ± 0.7	0.3 ± 0.2	3.8 ± 0.8	0.9 ± 0.2
Acala_SJ-2	Upland	1.8 ± 1.1	4.0 ± 0.6	0.8 ± 0.6	6.2 ± 1.1	1.4 ± 0.3
Acala_NemX	Upland	1.4 ± 0.6	3.4 ± 0.7	0.6 ± 0.4	6.6 ± 0.7	2.0 ± 0.2
13IN8227	Upland	0.8 ± 0.5	4.0 ± 0.6	0.3 ± 0.2	6.0 ± 0.9	1.4 ± 1.1
13IN8231	Upland	0.6 ± 0.4	4.4 ± 0.6	0.2 ± 0.2	5.8 ± 1.3	1.3 ± 0.2
13IN8236	Upland	0.4 ± 0.2	4.0 ± 0.6	0.2 ± 0.2	4.6 ± 1.2	1.6 ± 0.3
13IN8238	Upland	0.8 ± 0.6	4.0 ± 0.6	0.3 ± 0.2	4.0 ± 0.7	0.9 ± 0.3
13IN8239	Upland	0.6 ± 0.6	4.4 ± 0.6	0.2 ± 0.2	5.4 ± 0.5	1.0 ± 0.2
13IN8241	Upland	0.4 ± 0.4	4.6 ± 0.4	0.2 ± 0.2	5.4 ± 0.4	1.2 ± 0.1
13IN8242	Upland	0.6 ± 0.4	4.6 ± 0.4	0.0 ± 0.0	4.8 ± 0.6	1.1 ± 0.2
13IN8243	Upland	1.4 ± 0.7	4.2 ± 0.6	0.5 ± 0.3	5.6 ± 0.4	1.4 ± 0.3
13IN8247	Upland	0.4 ± 0.4	5.0 ± 0.0	0.0 ± 0.0	3.6 ± 1.0	1.1 ± 0.1
13IN8249	Upland	1.0 ± 0.6	4.2 ± 0.6	0.3 ± 0.2	4.8 ± 0.4	1.7 ± 0.5
13IN8250	Upland	0.6 ± 0.4	4.0 ± 0.6	0.3 ± 0.2	4.8 ± 1.0	1.2 ± 0.1
13IN8246	Upland	1.2 ± 0.6	4.2 ± 0.6	0.1 ± 0.1	7.2 ± 0.5	1.8 ± 0.5
13IN8251	Upland	0.8 ± 0.8	4.4 ± 0.6	0.2 ± 0.2	6.6 ± 0.7	1.4 ± 0.1
13IN8297	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	3.6 ± 1.1	0.9 ± 0.2
13IN8252	Upland	0.4 ± 0.2	4.4 ± 0.6	0.1 ± 0.1	6.4 ± 0.6	1.3 ± 0.2
13IN8254	Upland	0.2 ± 0.2	5.0 ± 0.0	0.0 ± 0.0	6.2 ± 0.7	1.4 ± 0.1
13IN8255	Upland	1.6 ± 0.8	4.4 ± 0.4	0.5 ± 0.3	6.0 ± 0.5	1.3 ± 0.2
13IN8258	Upland	0.8 ± 0.6	4.6 ± 0.4	0.1 ± 0.1	2.6 ± 0.7	0.6 ± 0.2
13IN8262	Upland	0.4 ± 0.2	3.8 ± 0.7	0.1 ± 0.1	2.4 ± 0.5	0.7 ± 0.1
13IN8263	Upland	0.4 ± 0.4	4.4 ± 0.6	0.3 ± 0.3	3.2 ± 0.4	0.9 ± 0.2
13IN8263 HWT*	Upland	0.4 ± 0.2	4.4 ± 0.6	0.1 ± 0.1	3.2 ± 0.3	1.0 ± 0.2
13IN8264	Upland	0.4 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	0.8 ± 0.4	0.2 ± 0.1
13IN8266	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	1.4 ± 0.5	0.3 ± 0.1
13IN8272	Upland	0.6 ± 0.6	4.6 ± 0.4	0.1 ± 0.1	5.2 ± 0.5	1.2 ± 0.2
13IN8273	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	3.4 ± 0.2	1.1 ± 0.1
13IN8274	Upland	0.2 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	6.0 ± 0.3	1.3 ± 0.3
13IN8275	Upland	0.2 ± 0.2	5.0 ± 0.0	0.0 ± 0.0	4.8 ± 0.8	1.3 ± 0.2
13IN8276	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	5.0 ± 1.0	1.0 ± 0.1
13IN8284	Upland	1.0 ± 0.5	3.6 ± 0.6	0.5 ± 0.3	2.4 ± 0.7	0.5 ± 0.1
13IN8285	Upland	0.4 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	4.0 ± 0.9	0.9 ± 0.2
13IN8285 HWT*	Upland	0.4 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	5.0 ± 0.9	1.3 ± 0.1
13IN8287	Upland	0.6 ± 0.4	3.8 ± 0.7	0.4 ± 0.3	2.8 ± 1.0	0.6 ± 0.3
13IN8291	Upland	1.0 ± 0.4	4.6 ± 0.4	0.3 ± 0.3	6.2 ± 1.0	1.5 ± 0.2
13IN8294	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	1.2 ± 0.7	0.1 ± 0.1
13IN8295	Upland	0.2 ± 0.2	4.4 ± 0.6	0.1 ± 0.1	5.6 ± 0.7	1.6 ± 0.4
MD51ne	Upland	1.6 ± 1.0	3.8 ± 0.7	0.4 ± 0.3	7.0 ± 0.6	1.7 ± 0.2
FM832	Upland	1.2 ± 0.8	3.8 ± 0.7	0.4 ± 0.3	7.0 ± 0.8	1.4 ± 0.1
13IN8302	Upland	0.2 ± 0.2	4.6 ± 0.4	0.1 ± 0.1	6.6 ± 0.5	1.6 ± 0.2
13IN8303	Upland	0.2 ± 0.2	4.4 ± 0.6	0.3 ± 0.3	5.0 ± 1.0	1.2 ± 0.2
13IN8312	Upland	0.4 ± 0.4	4.4 ± 0.6	0.2 ± 0.2	6.8 ± 0.5	1.8 ± 0.3
13IN8314	Upland	0.4 ± 0.2	3.8 ± 0.7	0.3 ± 0.2	7.2 ± 0.4	1.5 ± 0.4
13IN8319	Upland	1.4 ± 1.2	4.6 ± 0.4	0.3 ± 0.3	4.8 ± 1.2	1.1 ± 0.4
13IN8320	Upland	0.6 ± 0.4	4.4 ± 0.6	0.1 ± 0.1	5.2 ± 0.9	1.3 ± 0.4
13IN8321	Upland	0.2 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	4.2 ± 1.2	1.1 ± 0.1
13IN8336	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	6.4 ± 0.6	1.2 ± 0.2
13IN8338	Upland	0.8 ± 0.5	4.2 ± 0.5	0.3 ± 0.2	7.0 ± 0.5	1.4 ± 0.3
13IN8339	Upland	0.2 ± 0.2	4.6 ± 0.4	0.2 ± 0.2	5.4 ± 0.7	1.5 ± 0.2
13IN8342 HWT*	Upland	0.4 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	6.4 ± 0.2	2.0 ± 0.3
13IN8342	Upland	0.2 ± 0.2	4.4 ± 0.6	0.2 ± 0.2	5.8 ± 0.9	1.3 ± 0.2
13IN8344	Upland	1.4 ± 1.0	4.0 ± 0.6	0.5 ± 0.3	4.8 ± 1.2	0.9 ± 0.2
13IN8345	Upland	0.4 ± 0.2	3.8 ± 0.7	0.3 ± 0.2	2.6 ± 0.7	0.8 ± 0.3
13IN8347	Upland	0.4 ± 0.4	5.0 ± 0.0	0.0 ± 0.0	6.2 ± 0.8	1.4 ± 0.2
13IN8350	Upland	0.2 ± 0.2	4.4 ± 0.6	0.1 ± 0.1	3.2 ± 0.8	0.5 ± 0.1
13IN8356	Upland	2.2 ± 0.7	3.8 ± 0.5	0.7 ± 0.3	7.0 ± 0.4	1.3 ± 0.4
13IN8357	Upland	0.6 ± 0.4	4.2 ± 0.6	0.2 ± 0.1	5.4 ± 0.6	1.2 ± 0.1
13IN8359	Upland	0.4 ± 0.2	4.0 ± 0.6	0.4 ± 0.3	4.8 ± 0.5	1.2 ± 0.1
STV474	Upland	0.2 ± 0.2	5.0 ± 0.0	0.0 ± 0.0	7.2 ± 0.6	1.4 ± 0.2
13IN8368	Upland	0.4 ± 0.4	4.4 ± 0.6	0.1 ± 0.1	5.8 ± 0.9	1.3 ± 0.2
14TUN119.BULK	Upland	0.6 ± 0.6	4.4 ± 0.6	0.2 ± 0.2	3.0 ± 0.5	2.2 ± 0.5
14TUN219.BULK	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	3.4 ± 0.5	0.9 ± 0.2
NM67	Upland	1.2 ± 1.0	4.4 ± 0.6	0.4 ± 0.4	6.8 ± 0.7	1.2 ± 0.1
PHY72	Upland	0.4 ± 0.4	4.6 ± 0.4	0.2 ± 0.2	7.2 ± 0.6	1.7 ± 0.2
TM1	Upland	0.4 ± 0.2	5.0 ± 0.0	0.0 ± 0.0	3.8 ± 0.9	0.8 ± 0.1
FBCX-2	Upland	1.6 ± 1.1	4.0 ± 0.4	0.3 ± 0.3	5.8 ± 0.7	1.8 ± 0.4
Shorty	Upland	1.6 ± 0.9	4.2 ± 0.6	0.3 ± 0.2	6.0 ± 0.7	1.9 ± 0.4
Coker 312	Upland	1.0 ± 0.6	4.4 ± 0.6	0.2 ± 0.2	4.8 ± 0.5	0.9 ± 0.2
PSS-U77B	Upland	0.6 ± 0.4	4.4 ± 0.6	0.1 ± 0.1	6.0 ± 0.6	1.3 ± 0.2
Sel-1002	Upland	1.0 ± 0.8	4.6 ± 0.4	0.2 ± 0.2	7.0 ± 0.4	1.7 ± 0.3
Sel-1006	Upland	1.4 ± 1.0	3.8 ± 0.7	0.4 ± 0.3	7.2 ± 0.6	2.0 ± 0.4
Sel-1007	Upland	2.8 ± 1.2	3.4 ± 0.7	0.7 ± 0.3	7.2 ± 0.8	2.0 ± 0.5
Sel-1009	Upland	2.6 ± 1.3	4.0 ± 0.4	0.5 ± 0.2	7.2 ± 0.4	2.1 ± 0.6
Sel-1023	Upland	1.2 ± 0.5	2.6 ± 0.6	0.6 ± 0.3	7.2 ± 0.5	1.8 ± 0.4
Sel-1024	Upland	1.0 ± 0.4	3.6 ± 0.6	0.3 ± 0.2	7.0 ± 0.8	1.3 ± 0.1
Sel-1025	Upland	2.0 ± 1.5	4.4 ± 0.6	0.4 ± 0.4	7.0 ± 0.7	1.2 ± 0.1
Sel-1029	Upland	0.2 ± 0.2	4.4 ± 0.6	0.3 ± 0.3	6.4 ± 1.4	1.2 ± 0.1
Sel-1030	Upland	1.0 ± 0.8	4.0 ± 0.6	0.2 ± 0.2	5.2 ± 1.6	1.0 ± 0.3
Sel-1035	Upland	1.4 ± 0.9	4.4 ± 0.4	0.2 ± 0.2	7.0 ± 0.5	1.3 ± 0.2
Sel-1040	Upland	0.6 ± 0.4	4.0 ± 0.6	0.2 ± 0.2	6.8 ± 0.8	1.3 ± 0.3
Sel-1042	Upland	0.4 ± 0.2	4.6 ± 0.4	0.1 ± 0.1	6.4 ± 0.7	1.4 ± 0.1
Sel-1043	Upland	1.4 ± 1.4	4.4 ± 0.4	0.6 ± 0.3	7.2 ± 0.6	2.1 ± 0.7
Sel-1046	Upland	0.8 ± 0.8	4.6 ± 0.4	0.3 ± 0.3	7.2 ± 0.6	2.0 ± 0.4
Sel-1049	Upland	0.0 ± 0.0	5.0 ± 0.0	0.0 ± 0.0	7.0 ± 0.8	1.4 ± 0.1
Sel-1050	Upland	0.8 ± 0.8	4.6 ± 0.4	0.2 ± 0.2	7.4 ± 0.6	1.9 ± 0.5
Sel-1052	Upland	1.0 ± 0.6	4.6 ± 0.4	0.2 ± 0.2	7.0 ± 1.0	1.6 ± 0.3
Sel-1015	Pima	1.0 ± 0.4	3.6 ± 0.7	0.3 ± 0.2	7.2 ± 0.4	2.3 ± 0.3
Sel-1017	Pima	1.8 ± 1.2	4.4 ± 0.4	0.6 ± 0.5	7.0 ± 0.4	2.2 ± 0.3
Sel-1018	Pima	0.4 ± 0.2	5.0 ± 0.0	0.0 ± 0.0	5.8 ± 0.8	2.0 ± 0.3
SJ-10P20	Pima	1.6 ± 1.7	4.8 ± 0.2	0.4 ± 0.4	6.2 ± 0.4	2.0 ± 0.4
SJ-10P14	Pima	0.2 ± 0.2	4.6 ± 0.4	0.2 ± 0.2	6.2 ± 0.9	2.0 ± 0.4
DP744	Pima	0.4 ± 0.2	3.8 ± 0.7	0.3 ± 0.2	6.6 ± 0.5	1.7 ± 0.3
DP340	Pima	0.2 ± 0.2	4.8 ± 0.2	0.1 ± 0.1	6.6 ± 0.5	2.2 ± 0.6
P-3-79	Pima	1.0 ± 0.5	3.8 ± 0.6	0.3 ± 0.2	5.4 ± 0.6	1.6 ± 0.3
PS7	Pima	0.4 ± 0.2	3.8 ± 0.7	0.4 ± 0.3	5.4 ± 0.4	1.5 ± 0.2
PS6	Pima	0.4 ± 0.4	5.0 ± 0.0	0.0 ± 0.0	5.6 ± 0.8	1.9 ± 0.1

Note- Root rot was not observed in the non-inoculated cotton. Germplasm with an * are duplicates of genotypes that have seed with and without hot water seed treatments

These finding align with previous research by Stanton et al. [13], who similarly found no or low practical levels or weak resistance to R. solani in A-genome cotton lines (Gossypium arboreum L. and G. herbaceum L.) and by Poswal et al. and Jones et al. [4, 9] in the tetraploid (AD₂) G. hirsutum. These results are also consistent with previous studies [3, 4, 9], in which extensive screening was conducted of cotton lines, finding limited resistance to R. solani highlighting the challenges in identifying resistance germplasm.

Pest infestations caused by pathogens such as R. solani are related to a cotton seedling disease complex (CSDC), which occurs when an individual pathogen, or a complex of individual pathogens, infect the seed or the seedling stages of plant development [6]. The resistance CSDC is not well understood. Heritabilities in the narrow sense for R. solani ranges from 0.1 to 20.7%, with strong general combining ability effect. Resistance in cotton seedlings to R. solani seems to be polygenically inherited and conditioned by complex of minor genes in a F₂ generation [9]. Advances have been made at the gene expression level in which resistant plants have been found with at least two different mechanisms of resistance. One of the mechanisms is a constitutively activated response in Acala NemX which correlates with elevated levels of genes coding for disease resistance and receptor proteins linked to the Root-Knot Nematode (Meloidogyne incognita) -resistance that help plants to avoid a fitness penalty under disease pressure [7]. A second mechanism is through the fortification of the roots through an enhanced root phenylpropanoid metabolism in the resistant Pima-S6 cultivar that determines Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4)-resistance. Phenylpropanoid biosynthesis and metabolism categories correlated with the accumulation of secondary metabolites such as esculetin, a coumarin, an inhibitor of Fusarium’s growth in Pima-S6 roots [8].

The findings from our study and previous studies underscore the importance of continued screening of both historical and newer cotton germplasm to identify and harness genetic traits conferring enhanced resistance to R. solani. The lack of significant resistance identified in both historic and newer germplasm suggest a pressing need for further exploration and selection of novel sources of resistance within the vast genetic diversity of cotton germplasm.

Finally, although high levels of resistance were not observed among the germplasm that were evaluated in this study, it is important to note that in germplasm lines with higher stand counts and surviving plants (Table 2), the surviving plants could indicate some levels of resistance or weak resistance. This is especially true in the germplasm derived from crosses where some of the genetics may not be stable, and segregation at the highest rates is still occurring in newly developed progeny (F₂ through F₄ generation) (Table 1). Previous studies [4, 9, 14], have used and reported a method to improve resistance through a single or individual plant selection with several recurrent cycles of phenotypic selection under infested fields and/or artificial inoculation in the greenhouse. As used to increase and develop FOV4 resistant germplasm lines [15, 16], in future studies, surviving plants from germplasm lines such as 13IN8356, SEL1007, SEL1009, and SEL1025 with weak resistance may be used in breeding programs using this method to increase and develop R. solani resistant germplasm lines.

Limitations

This study focused exclusively on two cotton species, G. hirsutum (Upland) and G. barbadense (Pima), which limits the generalizability of the findings. Moving forward, screening efforts should be expanded to encompass a more comprehensive selection of cotton species. This expansion could involve incorporating additional species known for their distinct characteristics or exploring wild cotton relatives for novel resistance traits that could be harnessed through breeding programs. Additionally, there was a relatively smaller representation of Pima varieties (10 entries) compared to Upland cotton (85 entries). A broader inclusion of Pima varieties could have provided a more comprehensive understanding of the potential range of response to R. solani infection in Pima cotton.

Another limitation of this study that is important to note, is that for 19 of the Upland germplasm that were evaluated the non-inoculated controls had average stand counts of 50% or less indicating that low germination observed in the R. solani treatments were not entirely due to the infection by the pathogen (Table 2). Therefore, although these Upland germplasms also appear to be highly susceptible to R. solani, due to the low germination rates in the non-inoculated controls it is hard to make solid conclusions for these germplasm lines.

Another significant challenge this study faced is the high level of disease pressure, which prompted the utilization of the layer method as a strategy to mitigate disease impact. By employing the layer method, we aimed to minimize direct contact between the seeds and the soil, thereby providing the seedling a better chance of germinating before pathogen transmission and seedling infection. However, even using the layering method disease pressure was high and could have masked some of the variation in potential responses of the cotton germplasm to infection with R. solani.

Finally, it’s crucial to address the variability observed between replicates, which can be attributed to the timing of the experimental runs. Discrepancies in environmental conditions, such as temperature and humidity fluctuations, may have influenced seed germination rates and subsequent plant growth [12]. While efforts were made to standardize experimental conditions, inherent variability is inevitable in some greenhouse evaluations. This would be an even greater source of variability if we conducted field-based studies. Moreover, logistical constraints, including limited seed availability and resource allocation, influenced the number of experimental replicates conducted. Despite the potential benefit of conducting additional replicates under conditions conducive to germination, the observed trends did not warrant further runs. Balancing the need for robust statistical analysis with practical considerations such as seed availability and experimental resources is essential for optimizing research outcomes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

J.G. conducted the experiments as part of his graduate work for his M.S., wrote the majority of the manuscript, and read and approved manuscript. M.U. selected and provided the germplasm for the experiments, provided intellectual input to the study, and read, edited, and approved manuscript. R.B.H provided the greenhouse for the experiment, provided intellectual input to the study, and read, edited, and approved manuscript. M.L.E. supervised and supported M.S. student and experiments, provided help with the statistical analysis, helped write the manuscript, read, edited, and approved manuscript.

Funding

This research was partially funded by the California Cotton Alliance, California Cotton Ginners and Growers Association, Cotton Incorporated.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.