RESUMEN
Castor (Ricinus communis L.) oil is used in the manufacture of cosmetics, lubricants, plastics, pharmaceuticals, and soaps and is grown in more than 40 countries with India and China leading in oil production(Tunaru et al. 2012). In June 2021, a seedling rot disease was observed on castor cv. Zibi-5 in a plant nursery in Zhanjiang (21°17' N, 110°18' E), China. Initial symptoms on leaves and stems were water-soaked and dark green lesions that resulted in rapid rotting. Disease incidence was 25% and resulted in seedling death. White fungal mycelia developed on the rotting plant tissues. Leaves and stems were collected from 10 diseased plants, surface disinfected in 0.5% sodium hypochlorite and 75% ethyl alcohol, and tissue pieces placed in plates of potato dextrose agar (PDA) which were maintained at 28â. Hyphal tips from fungal mycelia that developed in the PDA plates were selected to establish pure cultures and three representative fungal isolates, designated RCC-1, RCC-2, and RCC-3, were selected for further study. The fungal isolates produced sporangiophores that were smooth, hyaline, aseptate, and apically swollen. Sporangiophores bore monosporous sporangiola that were broadly ellipsoidal, longitudinally coarsely striate, brown to dark brown, and measured 6.2 to 14.8 x 10.5 to 26.5 um (n=30). Sporangia contained few to many spores that were spherical, brown, and measured 59 to 150 um in diameter (n=20). Sporangiospores were ellipsoid, striate, and brown with multiple hyaline polar appendages and measured 6.6 to 12.3 x 10.6 to 25.5 um (n=30) in size. Based on these morphological characteristics, the fungus was identified as Choanephora cucurbitarum (Berk. & Ravenel) Thaxt. (Kirk, 1984). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and NL1/LR3 (Walther et al. 2013). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376748-OL376750, and LSU, OL763430-OL763432. BLAST analysis of these sequences revealed a 100% to 99% identity with the sequences (ITS, MG650194; 573/573, 573/573, and 573/573; LSU, AF157181; 673/676, 673/676, and 673/676) of C. cucurbitarum in GenBank. Pathogenicity tests, to fulfill Koch's postulates, were performed in a greenhouse with a temperature range of 24â to 30â and 80% relative humidity. Thirty-day-old cv. Zibi-5 castor plants were grown in pots and used for inoculation tests. Ten plants were inoculated by placing agar plugs with mycelia of fungal isolate RCC-1 on leaves or stems. Ten control plants were inoculated with agar plugs only and the test was repeated three times in total. Five days after inoculation, all plants, with either leaf or stem inoculations, became infected and began rotting. Symptom progression was consistent with that observed in the nursery and all control plants remained healthy. C. cucurbitarum was successfully reisolated from all inoculated plants and identified by morphological characteristics and by sequence analysis. This fungus is known to cause serious damage on a wide range of hosts (Liu et al. 2019) and previously was reported on castor in India (Shaw 1984) and Papua New Guinea (Peregrin and Ahmad 1982). We observed that the pathogen grows very rapidly and causes serious damage to castor seedlings, warranting further investigation on the epidemiology and control of this disease.
RESUMEN
Castor bean (Ricinus communis L.) is an important oil crop. Anthracnose lesions were observed on leaves of castor bean at the stage of budding and fruiting in field (21Ë17'51''N, 110Ë18'16''E), Zhanjiang, Guangdong Province, China in August 2019. The incidence rate was approximately 40% (n=600 investigated plants). Early symptoms were yellow spots appearing from the edge or the tip of the leaves. Later, the spots gradually expanded and became dark brown, which coalesced into larger irregular or circular lesions (Supplementary Figure 1). Seven diseased leaves were collected from seven plants. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A single-spore isolate (RLC-1) was used for further study. The colony of isolate RLC-1 on PDA was white to gray in color with cottony mycelia in 6 days at 28 °C. Conidia were one-celled, hyaline, cylindrical, clavate, obtuse at both ends and measured 14.2 to 18.5 µm × 3.8 to 5.5 µm (n =50). Appressoria were oval to irregular in shape, dark brown, and ranged from 7.3 to 10.5 µm × 5.7 to 6.5 µm (n = 20). Morphological characteristics of isolate RLC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate RLC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), and ACT (ACT-512F/ACT-783R) (Weir et al. 2012). Analysis of the ITS (accession no. MN880199), GAPDH (MN884048), and ACT (MN891766) sequences revealed a 99%-100% identity with the corresponding ITS (JX010250), GAPDH (KX578786), ACT (JX009541) sequences of C. siamense in GenBank. A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that clustered the isolate RLC-1 with C. siamense with the type strain ICMP 19118 (Supplementary Figure 2). Morphological characteristic and phylogenetic analysis identified the isolate RLC-1 associated with anthracnose of castor bean as C. siamense. Pathogenicity test was performed in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Twenty healthy plants of Zi Bi No. 5 castor bean (2 month old) were grown in pots with one plant in each pot. Inoculation was conducted on leaves with mycelial plugs of RLC-1 or agar plugs (as control). Three plugs were considered for each leaflet. Ten plants were used in each treatment (five for wounded inoculation and five for unwound inoculation). Anthracnose lesions as earlier were observed on the leaves after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all inoculated leaves was identical to the isolate RLC-1 by morphology and ITS analysis but not from control plants. C. siamense causes anthracnose on various plant hosts, including mango in Colombia (Pardo-De la Hoz et al. 2016) and Rosa chinensis in China (Feng et al. 2019) but not including castor bean. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on castor bean. Thus, this work provides a basis for focusing on the management of the disease in future.
RESUMEN
Castor bean (Ricinus communis L.) is an oil crop of significant economic importance in the industry and medicine. In August 2019, a branch dieback disease was observed on castor bean in a field in Zhanjiang (21.17°N, 110.18°E), China. The incidence rate was 35% (n=600 investigated plants). Symptoms were discoloration of leaves, branch dieback, and discoloration of internal stem tissues. The disease had spread to the whole branches and causing the plant to die. Seven diseased branches were collected from seven plants. Margins between healthy and diseased tissues were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Then, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Eighteen isolates were obtained (the isolate rate of 75%), which were the same fungus on the basis of morphological characteristics and molecular analysis of the internal transcribed spacer (ITS). A single representative isolate (RiB-1) was used for further study. The colony of RiB-1 was 5 cm in diameter on the 5th day on the PDA culture. The colony was greenish gray with an irregularly distributed and fluffy aerial mycelium, which turned black after 10 days. The mature conidia were 21.3-26.5 µm × 12.2-15.7 µm in size (n=100) and had two ovoid, dark brown cells with longitudinal striations. The morphological characteristics of the colonies were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). Three regions of the ITS, translation elongation factor (EF1-α), and ß-tubulin genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The resulting sequences were deposited in the GenBank under accession numbers MN759432 (ITS), MN719125 (EF1-α), and MN719128 (ß-tubulin). BLASTn analysis demonstrated that these sequences were 100% identical to the corresponding ITS (MK530052), EF1-α (MK423878), and ß-tubulin (MN172230) sequences of L. theobromae. Based on the morphological and molecular data, RiB-1 was determined as L. theobromae. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 °C to 30 °C. Ten healthy plants of Zi Bi No. 5 castor bean (1-month-old) were grown in pots with one plant in each pot. Five pots were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day cultures. Five additional pots treated with PDA plugs served as the controls. Inoculated stems were moisturized with sterile cotton for five days. The test was conducted three times. Disease symptoms, similar to those in the field, were observed on the inoculated plants two weeks after inoculation, and L. theobromae was 100% reisolated from the inoculated plants. The control plants remained symptomless, and reisolations were unsuccessful. These results consistent with Koch's postulates. L. theobromae (Lima et al. 1997) and L. hormozganensis (Fábio et al. 2018) had been reported to cause stem rot on castor bean in Brazil, but whether L. theobromae caused the branch dieback on castor bean in China has not been reported yet. Thus, this study is the first report of L. theobromae causing the branch dieback on castor bean in Zhanjiang, China. This study provides an important reference for the control of the disease.