Lines (258) from the USDA cotton collection were grown in soil that was artificially infested with Thielaviopsis basicola (causal agent of black root rot). A susceptible (‘Paymaster 2326 RR’) and a resistant (A20, Gossypium herbaceum) check were included in each test. Pots were grown for 25 days in a growth chamber at 19C and then the percent necrosis of the tap root was visually estimated. Lines that had levels of root necrosis similar to that of the resistant check included: TX1, TX 5, TX 15, TX 25, TX 27, TX 28, TX 29, TX 51, TX 68, TX 70, TX 83, TX 93, TX 96, TX 104, TX 111, TX 125, TX 129, TX 130, TX 140, TX 141, TX 143, TX 147, TX 151, TX 153, TX 163, TX 304, TX 320, TX 1867, and TX 2498. There were 58 lines that had an intermediate level of root necrosis and 171 lines that had levels of root necrosis similar to that of the susceptible check. Lines that tested resistant to black root rot originated from Ceara, Brazil (1), Chiapas, Mexico (2), Chiquimula, Guatamala (1), Jalapa, Guatamala (1), Jutiapa, Guatamala (8), Guatamala (3), Oaxaca, Mexico (3), Mexico (6), and St. Lucia (1).

Keywords: Gossypium hirsutum, seedling disease

Fungal pathogens of cotton seedlings include Rhizoctonia solani, Pythium spp., Fusarium spp., and Thielaviopsis basicola. Thielaviopsis basicola, the causal agent of black root rot, is primarily a post-emergence problem, where the lateral root system does not develop extensively, because of root necrosis. The fungus does not usually kill the seedlings, but does delay the development of lateral roots. Once the roots are able to slough off the necrotic root tissue, the lateral roots are able to form. The disease is most severe in weather where cool temperatures (≤ 21 C) persist (Bland et al., 1953; Rothrock, 1992). The fungus can also interact with the root-knot nematode, Meloidogyne incognita, in which case the damage to the roots is more extensive, and yield loss may increase (Walker et al., 1998; Walker et al., 1999; Walker et al., 2000).

Management of T. basicola is primarily achieved by planting under growing conditions that allow the plant to emerge and grow rapidly, or by crop rotation to nonhosts. Thielaviopsis basicola reproduces on a wide range of hosts (Johnson, 1916; Yarwood and Levkina, 1976), however, monocots are nonhosts. The primary infection propagule is similar to a chlamydospore, which survives well in the absence of a host. Therefore, in severe infestations, it may be necessary to rotate to nonhosts for a number of years. Chemical management with seed treatments can reduce the amount of root necrosis caused by T. basicola and increase yield (Kaufman and Wheeler, 1998). However, only partial control is achieved, and in moderate to severely infested fields, 100% root necrosis can occur even with fungicide seed treatments that are active against T. basicola. It is unlikely that a seed treatment will have sufficient systemic activity to protect a taproot. An in-furrow fungicide may be more effective in providing a zone of protection for roots, however, no fungicides have been labeled for that use in the United States.

Resistance to T. basicola has been identified and used in tobacco production (Haji et al., 2003). However, in most crops natural resistance has not been identified. Partial resistance was identified in G. arboreum (Wheeler et al., 1999), but no previous resistance of any strength has been found in G. hirsutum. It would greatly facilitate management of black root of cotton if there were a strong source of resistance available in G. hirsutum. The objective of this study was to screen some of the USDA cotton collection of G. hirsutum (http://www.ars-grin.gov/cgi-bin/npgs/html/tax_site_acc.pl?COT%20Gossypium%20hirsutum) for resistance to T. basicola.

METHODS AND MATERIALS

Seed was obtained from the USDA cotton germplasm collection and increased. Soil was autoclaved twice at 120C and 1.05 kg/cm² for 2.5 hours, before use. Thielaviopsis basicola was grown on carrot agar (Wheeler et al., 1999) for at least six weeks, and then the propagules were scraped off the plates and washed through a sieve with a 74-μm pore size. The inoculum density was adjusted to 500 clamydospores/cm³ soil. Soil (110 cm³) was added to a plastic Cone-Tainer (3.8 cm diameter, 14 cm long, Stewe & Sons, Corvallis, OR, USA), and a single seed was planted. There were 25 replications of each line planted for each test. In each test, a susceptible (‘Paymaster 2326 RR’, G. hirsutum) and a resistant line (A1 20, G. herbaceum) were included. Entries were arranged in a randomized complete block design and held in a growth chamber at 19C for 25 days. After 25 days, the roots were washed free of the soil and rated for percent necrosis. A total of 258 lines were screened.

Statistical analysis was done with PROC MIXED in SAS (SAS Institute, Cary, NC, USA). The fixed factor was line and replication was a random factor. The PDIFF test was used to determine if lines differed from the resistant or susceptible checks. There were an unequal number of observations between lines, since germination was very different between entries. Lines were considered different if P ≤ 0.05, however, it is unknown what effect the uneven number of observations of each entry had on the probability level.

RESULTS AND DISCUSSIONS

Lines that had levels of root necrosis similar to that of the resistant check (A1 20, G. herbaceum) included: TX1, TX 5, TX 15, TX 25, TX 27, TX 28, TX 29, TX 51, TX 68, TX 70, TX 83, TX 93, TX 96, TX 104, TX 111, TX 125, TX 129, TX 130, TX 140, TX 141, TX 143, TX 147, TX 151, TX 153, TX 163, TX 304, TX 320, TX 1867, and TX 2498 (Table 1). Locations with a high frequency of resistant lines included Ceara, Brazil (TX 2498) and St. Lucia (TX 1867), however, only one entry was tested from each of these sites. Locations with a moderate percentage of tested lines that were resistant were Jutiapa, Guatemala (8 of 36 were rated resistant) and Guerrero, Mexico (3 out of 21 were rated resistant) (Table 1). There were 58 lines that had an intermediate level of resistance to T. basicola (Table 2) where the percent of root necrosis was significantly less than the susceptible check, but higher than the resistant check. There were 171 lines that had a level of root necrosis similar to that of the susceptible check (data not shown).

The identification of resistance to T. basicola in tetraploid cotton should greatly facilitate the development of cotton lines with good agronomic properties and resistance to black root rot. Resistance to black root rot has been found in diploid cotton (i.e. A 20, Wheeler et al., 1999), however, it has proven difficult to move the resistance into tetraploid cotton. With a starting point of resistant tetraploid cotton, it should be possible to move the resistance into cotton lines with good agronomic properties much more rapidly than when starting with resistant diploid cotton. Resistant cultivars would be an excellent alternative to current methods of control.

REFERENCES

Bland, L. J., P. J. Leyendecker Jr., and R. M. Nakayama. 1953. Observations on black root rot

symptoms on cotton seedlings at different soil temperatures. Plant Dis. Rep. 37:473-476.

Haji, H. M., R. A. Brammail, and D. L. Van Hooren. 2003. Effect of the Nicotiana debneyi black

root rot resistance gene on the yield and quality characteristics of flue-cured tobacco in

Ontario. Canad. J. Plant Sci. 83:939-942.

Johnson, J. 1916. Host plants of Thielavia basicola. J. Agric. Res. 7:289-300.

Kaufman, H. W., T. A. Wheeler, R. Graves, G. Schuster, P. Kidd, and K. Siders. 1998. Large

plot performance of seedling disease seed treatment fungicides. 1:149-152. Proc.

Beltwide Cotton Conf., San Diego, CA 5-9 Jan. Natl. Cotton Council, Am., Memphis,

TN.

Rothrock, C. S. 1992. Influence of soil temperature, water, and texture on Thielaviopsis basicola

and black root rot on cotton. Phytopath. 82:1202-1206.

Walker, N. R ., T. L. Kirkpatrick, and C. S. Rothrock. 1998. Interaction between Meloidogyne

incognita and Thielaviopsis basicola on cotton (Gossypium hirsutum). J. Nematol.

30:415-422.

Walker, N. R., T. L. Kirkpatrick, and C. S. Rothrock. 1999. Effect of temperature on

histopathology of the interaction between Meloidogyne incognita and Thielaviopsis

basicola on cotton. Phytopath. 89:613-617.

Walker, N. R., T. L. Kirkpatrick, and C. S. Rothrock. 2000. Influence of Meloidogyne incognita

and Thielaviopsis basicola populations on early-season disease development and cotton

growth. Plant Dis. 84:449-453.

Wheeler, T. A., J. R. Gannaway, and K. Keating. 1999. Identification of resistance to

Thielaviopsis basicola in diploid cotton. Plant Dis. 83:831-833.

Yarwood, C. E. and L. M. Levkina. 1976. Crops favoring Thielaviopsis. Plant Dis. Rept. 60:347-

349.

Table 1. List of cultivars identified with resistance to Thielaviopsis basicola.

Entry	PI	# of plants rated	% Root necrosis	Rating/ Susceptible cultivarz	Rating/ Resistant cultivary	Collection area
TX1	153981	11	40	0.44	1.22	Mexico
TX5	153987	6	33	0.37	1.02	Guerrero, Mexico
TX15	154013	18	35	0.39	1.07	Mexico
TX25	154035	4	15	0.25	0.86	Mexico
TX27	154037	16	29	0.47	1.66	Chiapas, Mexico
TX28	154038	7	27	0.44	1.56	Mexico
TX29	154040	19	29	0.48	1.68	Mexico
TX51	154071	15	21	0.27	2.26	Chiapas, Mexico
TX68	153960	5	36	0.37	1.25	Guatemala
TX70	153964	17	41	0.42	1.41	Guatemala
TX83	153972	5	66	0.72	1.08	Guatemala
TX93	163654	11	68	0.74	1.11	Jutiapa, Guatemala
TX96	163665	11	57	0.62	0.94	Jutiapa, Guatemala
TX104	163676	11	68	0.74	1.11	Jalapa, Guatemala
TX111	163639	6	63	0.68	1.03	Jutiapa, Guatemala
TX125	165329	9	70	0.76	1.15	Mexico
TX129	165282	10	74	0.80	1.21	Oaxaca, Mexico
TX130	165296	11	64	0.70	1.05	Oaxaca, Mexico
TX140	163614	3	72	0.78	1.18	Jutiapa, Guatemala
TX141	163640	13	67	0.73	1.10	Jutiapa, Guatemala
TX143	163707	9	64	0.70	1.06	Chiquimula, Guatemala
TX147	165310	11	75	0.81	1.22	Oaxaca, Mexico
TX151	163633	10	65	0.71	1.07	Jutiapa, Guatemala
TX153	163653	7	69	0.75	1.14	Jutiapa, Guatemala
TX163	163641	3	58	0.63	0.96	Jutiapa, Guatemala
TX304	165366	3	20	0.27	8.89	Guerrero, Mexico
TX320	165385	3	20	0.27	8.89	Guerrero, Mexico
TX1867	530498	4	16	0.22	7.22	St. Lucia
TX2498	607803	12	14	0.19	6.11	Ceara, Brazil

^zThe susceptible check that was used in every test was ‘Paymaster 2326 RR’.

^yThe resistant check that was used in every test was A20 (Gossypium herbaceum).

Table 2. Identification of Gossypium hirsutum lines with weak resistance to Thielaviopsis basicola.

Entry	PI	# of plants rated	% Root necrosis	Rating/ Susceptible cultivarz	Rating/ Resistant cultivary	Collection area
TX3	153984	20	70	0.77	2.1	Guerrero, Mexico
TX6	153988	18	78	0.86	2.4	Puebla, Mexico
TX14	154011	41	77	0.85	2.3	Oaxaca, Mexico
TX17	154022	20	36	0.59	2.1	Mexico
TX22	154029	17	39	0.64	2.3	Chiapas, Mexico
TX67	154103	8	53	0.54	1.8	Chipas, Mexico
TX71	153965	15	49	0.50	1.7	Guatemala
TX75	153968	18	51	0.53	1.8	Guatemala
TX76	153968	8	51	0.53	1.8	Guatemala
TX77	153969	10	69	0.71	2.4	Guatemala
TX78	153969	10	52	0.54	1.8	Guatemala
TX184	163642	8	57	0.67	3.6	Jutiapa, Guatemala
TX187	163723	12	60	0.71	3.7	Zacapa, Guatemala
TX198	163655	11	37	0.44	2.3	Jutiapa, Guatemala
TX206	165368	14	61	0.72	3.8	Guerrero, Mexico
TX215	163637	12	61	0.72	3.8	Jutiapa, Guatemala
TX216	163649	16	63	0.75	4.0	Jutiapa, Guatemala
TX220	163683	19	43	0.50	2.7	Jalapa, Guatemala
TX221	163706	13	56	0.66	3.5	Chiquimula, Guatemala
TX228	163672	60	54	0.64	3.4	Jalapa, Guatemala
TX236	163650	12	36	0.43	2.3	Jutiapa, Guatemala
TX243	165324	14	58	0.68	3.6	Oaxaca, Mexico
TX247	163631	13	37	0.44	2.3	Jutiapa, Guatemala
TX248	163673	2	60	0.71	3.8	Jalapa, Guatemala
TX256	165245	10	53	0.63	3.3	Oaxaca, Mexico
TX257	165253	7	55	0.65	3.5	Oaxaca, Mexico
TX259	165267	13	59	0.70	3.7	Oaxaca, Mexico
TX264	165302	10	58	0.69	3.6	Oaxaca, Mexico
TX277	165249	4	58	0.68	3.6	Oaxaca, Mexico
TX280	165292	11	46	0.63	20.6	Oaxaca, Mexico
TX281	165299	18	46	0.63	20.5	Oaxaca, Mexico
TX282	165306	16	53	0.72	23.5	Oaxaca, Mexico
TX285	165251	13	45	0.62	20.0	Oaxaca, Mexico
TX292	165230	13	45	0.62	20.2	Oaxaca, Mexico
TX295	165252	13	46	0.63	20.3	Oaxaca, Mexico
TX296	165266	11	39	0.53	17.2	Oaxaca, Mexico
TX297	165273	9	43	0.59	19.3	Oaxaca, Mexico
TX298	165280	17	34	0.46	15.0	Oaxaca, Mexico
TX301	165301	18	47	0.65	21.0	Oaxaca, Mexico
TX303	165352	10	46	0.63	20.4	Oaxaca, Mexico
TX305	165376	6	43	0.58	18.9	Guerrero, Mexico
TX311	165370	4	35	0.48	15.6	Guerrero, Mexico
TX325	165393	14	36	0.49	16.0	Guerrero, Mexico
TX1148	273895	20	38	0.52	16.9	Kefa, Ethiopia
TX1533	530164	13	49	0.67	21.7	Martinique
TX1585	530216	15	41	0.57	18.4	Haiti
TX1612	530243	14	38	0.52	17.0	Guadeloupe
TX1614	530245	12	47	0.64	20.9	Guadeloupe
TX1620	530251	5	40	0.55	17. 8	Guadeloupe
TX1824	530455	15	24	0.32	10.5	Dominican Republic
TX1841	530472	4	45	0.62	20.0	Desirade, Guadeloupe
TX1862	530493	12	50	0.68	22.0	Guadeloupe
TX2115	478759	16	45	0.62	20.1	Australia
TX2214	501394	14	52	0.71	23.2	Netherlands Antilles
TX2237	501417	13	31	0.43	13.8	Jamaica
TX2297	501477	13	38	0.52	16.8	Puerto Rico
TX2852		3	43	0.59	19.3

^zThe susceptible check that was used in every test was ‘Paymaster 2326 RR’.

^yThe resistant check that was used in every test was A20 (Gossypium herbaceum).