Fungi associated with boll and lint rot of cotton in

Archives of Phytopathology and Plant Protection, 2013 http://dx.doi.org/10.1080/03235408.2013.765135

Fungi associated with boll and lint rot of cotton in Southern Khorasan province of Iran M.R. Mirzaeea*, A. Heydarib, R. Zareb, L. Naraghib, F. Sabzalic and M. Hasheminasabc a

Agricultural and Natural Resources, Research Center of Southern Khorasan, Birjand, Iran; Iranian Research Institute of Plant Protection, Tehran, Iran; cAgricultural Management of Nehbandan, Nehbandan, Iran b

(Received 5 January 2013; final version received 6 January 2013) During surveys of cotton fields since 2009 in the Southern Khorasan province of Iran, symptomatic samples with lint and boll rot disease signs were collected and cultured on potato dextrose, malt extract agar and PARPH media. Several fungal isolates were obtained and identified. Among them, we found Penicillium expansum which to our knowledge is reported as a new pathogen of cotton worldwide; Fusarium semitectum and Nigrospora oryzae (associated to mite Siteroptes sp.) were also recorded as lint or boll rot pathogens for the first time from Iran. Disease symptoms and fungal characteristics of Exserohilum rostratum, which has been reported briefly as a new causal agent of boll and lint rot disease, are illustrated and presented. In addition, Aspergillus niger and Rhizopus spp. were also isolated frequently from lint parts of cotton prior to harvesting. There was a positive correlation between disease occurrence and spiny cotton bollworm damage. The pathogenicity test was characterised by inoculating the attached and detached bolls of cotton plants. Keywords: Gossypium hirsutum; etiology; cotton diseases; new pathogens

Introduction Cotton (Gossypium hirsutum L), is an important cash crop, grown in many countries around the world including Iran (Mansoori and Hamdollahzadeh 1995) and is planted in approximately 200,000 ha in more than 15 provinces of Iran (Heydari et al. 2007). Boll rots cause significant losses by reducing yields, discoloration and decreasing the quality of the lint, and by infecting the seed with pathogenic organisms which may cause seedling blights later (Halisky et al. 1961). A few fungal taxa including Alternaria spp., Ascochyta gossypii, Aspergillus flavus, Colletotrichum spp., Fusarium spp., Rhizoctonia solani and Phytophthora nicotianae are known as primary causal agents of cotton boll and lint rot worldwide that are capable of entering through openings or directly to intact bolls (Allen and West 1986; Kirkpatrick and Rothrock 2001). Meanwhile, many agents are opportunistic wound pathogens that cause disease following insect damage or other wounds to bolls such as Alternaria, Curvularia, Rhizopus and Penicillium (Kirkpatrick and Rothrock 2001). Although, mycoflora of cotton seed and seed-borne fungi of some cotton cultivars have been investigated previously, there is no report on the causal agents of the cotton boll rot disease in Iran (Mansoori 1993; Soleimani et al. 1993). Average loss caused by cotton boll rot has been estimated as nearly 5% in Golestan province, Northern Iran. *Corresponding author. Email: [email protected] Ó 2013 Taylor & Francis

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Fusarium sp. and Aspergillus sp. have been reported as the causal agents of cotton boll rot in Golestan province (Houshyarfard and Miri 2010). The aim of this study was to isolate and identify the causal agents of boll and lint disease of cotton in a semi-arid region of Iran, in Southern Khorasan province. An abstract based on a part of this study has been published introducing Exserohilum rostratum as a new causal agent of boll and lint rot disease of cotton (Mirzaee, Zare, Heydari et al. 2010). Materials and methods A comprehensive survey on boll and lint rot disease was conducted in cotton fields of Southern Khorasan province during 2009–2011 in order to determine the fungal pathogens involved (Table 1). The specimens were taken at random and samples were immediately taken to the laboratory in labelled plastic bags for future studies. Symptomatic cotton tissues were excised, surface-disinfected by immersing in 0.5% (v/v) sodium hypochlorite solution for 90 s, rinsed in sterile distilled water, placed on potato dextrose agar (PDA), malt extract agar (MEA), and semi-selective PARPH medium and incubated at 26 °C in the dark. The frequency percentage of fungal taxa was calculated as the percentage of pieces showing fungal growth including total number of collections of particular taxon encountered/total number samples examined 100 (Pinruan et al. 2007). Fungi were stained in 3% KOH plus cotton blue and examined under light microscope (Rubini et al. 2005). Single-spore cultures were used to perform pathogenicity tests from pure cultures grown for 10–15 days in Petri dishes containing one-quarterstrength PDA (1/4PDA; potato 50 g; dextrose 5 g and agar 20 g) (Mirzaee, Zare, and Azari 2010; Segalin and Reis 2010). Attached and detached bolls of cotton plants, cv. Varamin, inoculated by either 5-mm diameter fungal mycelium-containing agar plugs or with injection of conidial suspension (105 conidia/mL), were wrapped with moist cotton and parafilm and placed into closed, translucent, plastic bags to prevent drying. Control bolls were treated with sterile distilled water or agar plugs free from fungal mycelium. Disease evaluation was done every week, observing symptoms and disease progress for three weeks after inoculation and in order to confirm Koch’s postulates, fungi were re-isolated and identified. Table 1. Distribution and average frequency percentage of fungal taxa causing cotton lint and boll rot disease in Southern Khorasan, Iran. Fungus Aspergillus niger Cladosporium cladosporioides Exserohilum rostratum Fusarium cf. semitectum Nigrospora oryzae Penicillium expansum Rhizopus spp.

Average frequency percentage 45.6

Locations

8.4

Nehbandan (Chahdashi) Boshrouye, Mohammadiah, Birjand (Siyujan) Boshrouye, Mohammadiah, Birjand (Siyujan)

3.00

Nehbandan (Chahdashi)

6.8

Nehbandan (Chahdashi), Mohammadiah

5.6 0.2 30.4

Nehbandan (Chahdashi), Birjand (Siyujan) Boshrouye Nehbandan (Chahdashi) Boshrouye, Mohammadiah, Birjand (Siyujan)

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Results and discussion During this survey on cotton fields since summer 2009, in the Southern Khorasan province, various rot symptoms were observed on lint and bolls of cotton. Several pathogenic fungal taxa were isolated from the symptomatic tissues as described below: Exserohilum rostratum This fungus as a new causal agent of boll and lint rot disease of cotton was briefly recorded with low frequency percentage (Table 1). Disease symptoms and fungal characteristics are illustrated and presented. The fungus was identified by its characteristic velvety, dark-brown colonies, simple conidiophores, up to 200 μm straight to slightly curved conidia, 12.5–20 27.5– 105 μm distinctly protuberant hilum and bipolar germination (Sivanesan 1987; Figures 1 and 2). Symptoms included internal lint rot within multiple locules and external mature boll damage leading to complete olive to dark-brown decay of bolls associated with spiny bollworm (Earias insulana Boisduval) damage (Figure 3). Nigrospora oryzae (Berk. & Broome) Petch Symptoms caused by this fungus appeared as internal rotted boll and lint rot without extensive external damage in boll-opening period. The affected lint was compact and grey, leading to the characteristic “grey lock” symptom. Colonies on PDA appeared brown or black, mycelium immersed or partly superficial. Conidiophores were branched, flexuous and colourless to brown, smooth, 4–7 μm thick. Conidiogenous cells were monoblastic, solitary and 5–10 5–7 μm in diameter.

Figure 1.

E. rostratum, conidia with dark delimitation bands at both ends and an obvious hilum.

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Figure 2.

E. rostratum, conidiophore and conidia.

Figure 3.

Lint rot symptoms on internal boll caused by E. rostratum.

Conidia were single, smooth, spherical to broadly ellipsoidal, dark brown to black, shining, one celled and 17–20 μm in diameter (Ellis 1971; Palmateer et al. 2003; Figure 4).


Figure 4.

N. oryzae, hyphae and conidia.

Figure 5.

Representation of the Nigrospora oryzae associated to mite Siteroptes sp. on PDA.

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Association of the fungus with a mite, Siteroptes sp. was observed in media cultures as abundant colonies (Figure 5). Siteroptes reniformis serves as a vector of this fungus and there is a type of symbiotic interaction between them. Therefore, the occurrence of the disease may be dependent on the mite association (Laemmlen and Hall 1973). This, to our knowledge, is the first report of this fungus causing lint rot disease on cotton in association with Siteroptes mite in Iran. Fusarium cf. semitectum Berk. & Ravenel Fusarium semitectum has been reported to causes significant damage to cotton by inciting boll rot disease in India and Columbia under hot and humid conditions which indicates that seed inoculum potential may be related to this disease (Costa et al. 2005). Disease symptoms are observed after October rainfall as dried boll rot with cream to pinkish discoloration. The fungus typically colonises the surface of the carpel and boll and produces orange masses (Figure 6). Tan colony colour, looking powdery, chlamydospores in chain and smooth but not very abundant, monophialides and polyblastic (rare), no micro, curved macro with pointed apical cell, lack of mesoconidia on SNA. Three to five septate macroconidia, 38 4 μm in diam. were produced in sporodochia on carnation leaf agar (Figure 7). On PDA, cultures produce light orange sporodochia and abundant dense aerial mycelia that becomes beige or brown with age. Based on morphological and cultural characteristics, the fungus was identified as F. cf. semitectum Berk. & Ravenel (Leslie and Summerell 2006). Fusarium roseum,

Figure 6.

Boll-rot symptoms on a cotton boll caused by F. cf. semitectum.


Figure 7.

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Chlamydospores and macroconidia of F. cf. semitectum.

F. oxysporum, F. solani, F. lateritectum, F. moniliforme and F. semitectum have previously been reported as the causal agents of cotton boll rot disease (Kirkpatrick and Rothrock 2001). This is thought to be the first report of F. cf. semitectum causing cotton boll rot in Iran. Penicillium expansum Symptoms included internal lint and boll rot in green colour (green rot), associated with spiny bollworm damage, collected in October 2009 (Figure 8). Colonies on MEA plane with irregular growth and velutinose texture, sometimes becoming crustose; colour yellow green to blue green; pale to yellow brown reverse. Conidiophores hyaline, single or in fascicles, appressed, stipes usually smooth-walled, occasionally very finely rough-walled, terverticillate, less commonly biverticillate; rami cylindrical, 15–25 3–4 μm; metulae more or less cylindrical, 10–15 3–4 μm; phialides cylindrical with a short but distinct neck, 9–12 2.5–4 μm; conidia smooth-walled, ellipsoidal, 3–4 2.5–3 μm diamete (Kim et al. 2007; Kozakiewicz 1995). This fungus is capable of entering through wounds and has been reported as the causal agent of blue mould disease of wheat and sorghum seeds and fruit soft rot of apple, pears, cherries and pomegranate with carcinogenic metabolites producing potential (Farr and Rossman 2012; Palou et al. 2010; Rosenberger 1990). This, to our knowledge, is the first report of this fungus causing a disease on cotton.

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Figure 8.

Green lint rot disease caused by P. expansum.

Aspergillus niger, Rhizopus stolonifer, R. nigricans and Cladosporium cladosporioides were also isolated from all surveyed fields near harvest time. Aspergillus spp. and Rhizopus spp. have been reported as more widespread pathogens in southern California because the higher summer temperatures favour their development there. The most frequency of these fungi was recorded from Nehbandan region with higher summer temperatures (data unpublished). The symptoms of A. niger and Rhizopus spp. were typically as grey to black lint rot with conidial masses and together with spiny cotton bollworm damage. Pathogenicity test was positive on mature bolls only and symptom progress was slow. A. niger was also isolated from immature bolls with abscission potential. A. niger is a known producer of mycotoxins (Frisvad et al. 2007; Soares et al. 2013). Due to importance of cotton seed and its products in both animal and human nutrition and the unusual hazards associated with the aflatoxins (Simpson et al. 1973), it is necessary to make further observation and investigations. The symptoms progress induced by Cladosporium cladosporioides was more slower than the disease symptoms induced by A. niger and Rhizopus spp. According to previous studies, some fungal pathogens such as Diplodia, Fusarium and Rhizopus species can be transmitted by the spiny bollworm, pink bollworm and common bollworm (Halisky et al. 1961; Tu 1969). To prevent losses from boll rot disease, control of rank growth and weeds, bottom defoliation, skip row planting, plantation of cotton plants with okra type leaves and management of insects that damage cotton bolls have been recommended (Andries et al. 1969; Halisky et al. 1961; Houshyarfard and Miri 2010).


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The disease incidence in Boshrouye region with optimal cultural management was significantly less than other regions. On the other hand, early plantation of cotton seeds (late April rather than mid or late June) had decreased spiny cotton bollworm damage severity, and subsequently, cotton boll disease and did not subject cotton boll to rainfall at fall. We therefore recommend early plantation using fast ripping cultivars such as cv. Khordad. Sun burning is a significant disorder of bolls in some cotton fields of the province which has mostly been observed in the margins of the fields. Association of boll and lint rot symptoms was not observed with this disorder damage. Although in some cases, the association was observed with Aspergillus sp. damage, because of subjecting the fungus to the opened bolls. Boll rot disease symptoms were consistently associated with spiny bollworm indicating that this pest serves as a vector of pathogens causing cotton boll and lint disease and boll penetration does not have a role in disease infections. References Allen SJ, West KL. 1986. Phytophthora boll rot of cotton. Australas Plant Pathol. 15:34. Andries JA, Jones JE, Sloane LW, Marshall JG. 1969. Effects of okra leaf shape on boll rot, yield, and other important characters of upland cotton Gossypium hirsutum L. Crop Sci. 9:705–910. Costa MLN, Dhingra OD, DA Silva JL. 2005. Influence of internal seedborne Fusarium semitectum on cotton seedlings. Fitopatol Bras. 30:183–186. Ellis MB. 1971. Dematiaceous hyphomycetes. Kew: C.M.I. Farr DF, Rossman AY. 2012. Fungal databases, systematic botany & mycology laboratory. ARS, USDA. Available from http://nt.ars-grin.gov/fungaldatabases Frisvad JC, Smedsgaard J, Samson RA, Larsen TO, Thrane U. 2007. Fumonisin B2 production by Aspergillus niger. J Agri Food Chem. 55:9727–9732. Halisky PM, Schnathorst WC, Erwin DC. 1961. Distribution and control of cotton boll rots in California cotton growing areas. California Agric Exp Station. 15:6–7. Heydari A, Ahmadi A, Sarkari S, Karbalayi KH, Delghandi M. 2007. Study on the role of common weeds in survival of Verticillium dahliae the causal agent of cotton wilt disease. Pak J Biol Sci. 10:3910–3914. Houshyarfard M, Miri AA. 2010. Effect of skip-row planting pattern on yield, yield components, lint quality and diseases infection in two cotton (Gossypium hirsutum L.) genotype. Seed Plant Product J. 26: 301–316. Kim WK, Sang HK, Woo SK, Park MS, Paul NC, Yu SH. 2007. Six species of Penicillium associated with blue mold of grape. Mycobiology. 35:180–185. Kirkpatrick TL, Rothrock CS, editors. 2001. Compendium of cotton diseases., 2nd ed. St. Paul (MN): APS Press. Kozakiewicz Z. 1995. Penicillium expansum. IMI Descriptions Fungi Bacteria. 126:1258. Laemmlen FF, Hall DH. 1973. Interdependence of a mite, Siteroptes reniformis, and a Fungus, Nigrospora oryzae, in the Nigrospora lint rot of cotton. Phytopathology. 63:308–315. Leslie JF, Summerell BA. 2006. The Fusarium laboratory manual. Ames, IA: Blackwell. Mansoori B. 1993. Seedborne fungi of cotton in Iranian 11th Plant Protection Congress, Rasht, Iran. Mansoori B, Hamdollahzadeh A. 1995. Seed rot and seedling diseases of cotton in Gorgan and Gonbad. Appl Entomol Phytopathol. 62:80–83. Mirzaee MR, Zare R, Azari A. 2010. A new leaf and sheath brown spot of foxtail millet caused by Bipolaris australiensis. Austral Plant Dis Notes. 5:19–20. Mirzaee MR, Zare R, Heydari A, Naraghi L, Mohammadi M. 2010. First report of boll and lint rot disease of cotton caused by Exserohilum rostratum in Iran. J Plant Pathol. 4:S4–117. Palmateer AJ, McLean KS, Santen E, Morgan-Jones G. 2003. Occurrence of Nigrospora lint rot caused by Nigrospora oryzae on cotton in Alabama. Plant Dis. 87:873. Palou L, Guardado A, Montesinos-Herrero C. 2010. First report of Penicillium spp. and Pilidiella granati causing postharvest fruit rot of pomegranate in Spain. New Dis. Rep. 22, 21.

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M.R. Mirzaee et al.

Pinruan U, Hyde KD, Lumyong S, McKenzie EHC, Jones EBG. 2007. Occurrence of fungi on tissues of the peat swamp palm Licuala longicalycata. Fungal Diversity. 25:157–173. Rosenberger, DA. 1990. Blue mold. In: Jones AL, Aldwinckle HS, editors. Compendium of apple and pear diseases. St. Paul, MN: American Phytopathological Society Press. p. 54–55. Rubini MR, Silva-Ribeiro RT, Pomella AWV, Maki CS, Araujo WL, dos Santos DR, Azevedo JL. 2005. Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of ‘witches’ broom disease. Int J Biol Sci. 1:24–33. Segalin M, Reis EM. 2010. Semi-selective medium for Fusarium graminearum detection in seed samples. Summa Phytopathol. 36:338–341. Simpson ME, Marsh PB, Merola GV, Ferrettirj, Filsinger EC. 1973. Fungi that infect cottonseeds before harvest. Appl Microbiol. 26:608–613. Sivanesan A. 1987. Graminicolous species of Bipolaris, Curvularia, Drechslera, Exserohilum and their teleomorphs. Mycol Pap. 158:1–261. Soares C, Calado T, Venâncio A. 2013. Mycotoxin production by Aspergillus niger aggregate isolated from harvested maize in three Portuguese regions. Revista Iberoamericana de Micologia. 30:9-13. Soleimani M, Hedjarude GH, Zad J. 1993. Survey on mycoflora of cotton seed in Iran. Iran J Plant Pathol. 29:132–139. Tu CC. 1969. Studies on the role of insects in cotton boll black rot occurrence. J Taiwan Agric Res. 18:78–82.