FIRST REPORT OF Lichtheimia sp. AND Neurospora sitophila CAUSING CASSAVA TUBER ROT IN VIET NAM

FIRST REPORT OF Lichtheimia sp. AND Neurospora sitophila CAUSING CASSAVA TUBER ROT IN VIET NAM

Pham Viet Cuong

Assoc. Prof., Ph.D, Mientrung Institute for Scientific Research, Vietnam Academy of Science and Technology, Viet Nam, Ha Noi

Tran Thi Hong

Assoc. Prof., Ph.D, Mientrung Institute for Scientific Research, Vietnam Academy of Science and Technology, Viet Nam, Ha Noi

ABSTRACT

The cassava (Manihot esculenta Crantz) has an important role in the world economy, especially in Vietnam. However, in recent years, cassava has suffered from some serious diseases; amongst them, tuber rot is the most important cause of loss of cassava production, causing a lot of damage to the farmers and this is mainly due to fungal diseases. In May 2019, fungi were isolated from tubers of cassava plants showing typical tuber rot disease collected from Tay Ninh province, a major cassava cultivating region of Vietnam. Lichtheimia sp. (VTCC 930027) and Neurospora sitophila (VCCM 34008) were identified as causes for tuber rot based on morphological characteristics. The identification of one fungus was confirmed by the analysis of internal transcribed spacer region (ITS). Pathogenicity tests were conducted to satisfied Koch’s postulates. This is the first report of cassava tuber rot caused by Lichtheimia sp. and Neurospora sitophila in Vietnam. This research helps to better understand fungi that are causing cassava tuber rot disease in Vietnam as well as in the world and will help design strategies to manage the disease of this crop.

Keywords: Cassava tuber rot disease; Lichtheimia; Neurospora sitophila; pathogenicity.

I. INTRODUCTION

Cassava (Manihot esculenta Crantz) is a perennial root food crop, belongs to the botanical family Euphorbiaceous and is one of the world’s most important food crops [13]. However, the mass production of cassava also presents major risks, including fungi disease outbreaks, and depletion of soil nutrients [5]. According to reports by Sankar et al. (2014), rot in tubers of cassava has been detected since 1999, in some cassava growing areas of India and the disease has spread to the other regions in Kollam, Kottayam of Kerala (since 2005), accounts for yield losses up to 50-70% [14]. Infected tuber exhibits brown lesions with watery exudates and foul smell, making it useless for further use. At the Tamil Nadu and China, cassava tuber rot with similar symptoms was reported by Johnson and Palaniswami (1999) [8] and Guo et al. (2012) [6]. Both publications indicated that Phytophthora palmivora was the causative agent of the disease. In addition, some other fungi such as Scytalidium lignicola, Fomes lignosus, ... can cause root rot in seedling, tuber rot of mature plants in poorly drained soils or a heavy rainy season [2, 11].

There is scarcity of information so far on the tuber rot diseases of cassava in Vietnam as well as in the world. This marks the first report of cassava root rot caused by Lichtheimia sp. and Neurospora sitophila in Vietnam. This research helps to better understand fungi that are causing tuber rot disease of the cassava. The obtained results will help design strategies to manage the disease of this crop, making a safe and sustainable agriculture in Vietnam as well as in the world.

II. MATERIALS AND METHODS

Isolation of fungal pathogens

In May 2019, cassava tuber rot at Tan Chau district, Tay Ninh province, Vietnam were found with symptoms such as tubers soft rot, infected tissues appeared milky, gray to brown discoloration and smelled fermented silage (Fig. 1). Tuber samples with rot were packed in a cooler box, and transported to the laboratory of Nghia Do Centre for Molecular Biology at Hanoi, Vietnam within 12 hours for analysis.

Figure 1. Symptoms of cassava tuber rot disease in Tan Chau, Tay Ninh province

Tuber samples with rot were first washed in sterile distilled water and surface sterilized in 70% ethanol for 2 min, rinsed twice in sterile distilled water and allowed to dry on sterile tissue paper. Following incubation in Petri dishes for 2 - 3 days at 25 - 28 °C, mycelia grown from tuber fragments were transferred and maintained on PDA medium (agar 15 g/L, dextrose 20 g/L,  potato extract 4 g/L) [9, 14, 16].

Pathogenicity tests

Pathogenicity of the isolates was determined by artificial inoculation method as described by Sankar et al. (2012) [15] and Kibemo (2017) [9].

For the in vitro tests, fresh stems and tubers from 8-12 month old cassava seedling were washed with tap water followed by surface sterilized using sodium hypochlorite (NaOCl) for 1 min, rinsed with sterile distilled water and blot dried. The stems and tubers were drilled on both edges (upper and lower) to a depth of 1 cm, cut with a sterile cork borer. 20 µL of each isolate (7 days old on the PDA medium, approximately 2 x 103 CFU/mL) was inoculated into the hole and sealed with the stem’s piece, tuber’s piece removed from the hole, then points of inoculation were sealed with parfilm. For control, stems and tubers were inoculated with sterile distilled water. Each experiment was repeated thrice with three replicates. All the stems were placed in the flasks at 28 oC for 7 days.

In the in vivo tests, wounds were made on the surface of the root of healthy seedlings carefully by using sterilized knife. Then, the root of healthy seedlings was inoculated with 15 mL of conidia suspension (10⁵ conidia/mL) of each fungal isolates in plastic containers. Control seedlings were inoculated with sterile distilled water. Each experiment was repeated thrice with three replicates. The inoculated seedlings were kept under shade on open air environment for 1 month.

Morphological characterization

The pure fungal strains were transferred to PDA and MEA medium to determine colony characteristics. Observe the conidia by an electron microscope. Using Illustrated Genera of Imperfect Fungi for the mycelium to identify species.

Identification of fungal pathogens

DNA of the isolate was extracted according to Mishra et al. (2014) [7]. The rDNA fractions of the fungus were amplified with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) [17]. The PCR cycling parameters are as follows: an initial denaturation at 94 oC for 3 minutes, followed by 30 cycles of denaturation at 94 oC for 30 seconds, annealing at 52 ^oC for 30 seconds, and 72 ^oC for 1 minute, and a final extension at 72 ^oC for 10 minutes. PCR products are stored at 8 ^oC. The nucleotide sequence was analysed in NCBI database (BLAST).

III. RESULTS AND DISCUSSION

A total of six strains of fungi were isolated from six samples of cassava tuber rot at Tan Chau district, Tay Ninh province, Vietnam. For all isolates, single hyphal tip cultures were produced under the stereomicroscopefrom the margins of fresh cultures on agar medium (WA), next, the fungal samples were purified on PDA and kept freeze-dried at -80 ^oC. These fungal isolates were assessed for their pathogenic ability on healthy stem, tubers, and seedlings of 94 and 419 variety of cassava in Vietnam.

Results of pathogenicity test showed that out of six fungal isolates, 2 strains exhibited very strong tuber rot, namely TAN2PD11 and TAN3PD12. The diseases symptoms were similar on both 94 and 419 cassava cultivars. The symptom started developing on the stems and tubers after 7 days of inoculation, on the seedlings after 10 days of inoculation. Disease symptom was typical and similar to those symptoms in field conditions. No symptoms were observed in the controls (Fig. 2 and Fig. 3).

Figure 2. Pathogenicity test on stems, tubes and seedlings from healthy cassava plants. A, D: Symptoms of stem rot caused by TAN3PD12 and control, respectively. B, E: Symptoms of tuber rot caused by TAN3PD12 and control, respectively. C, F: Symptoms of seedlings rot caused by TAN3PD12 and control, respectively

Figure 3. Pathogenicity test on stems, tubes and seedlings from healthy cassava plants. A, D: Symptoms of stem rot caused by TAN2PD11 and control, respectively. B, E: Symptoms of tuber rot caused by TAN2PD11 and control, respectively. C, F: Symptoms of seedlings rot caused by TAN2PD11 and control, respectively

Based on morphological characteristics, TAN3PD12 and TAN2PD11 were identified as Neurospora sitophila (Fig. 4) and Lichtheimia sp. (Fig. 5). ITS amplified region upon sequencing yielded about 508 and 513 bp, respectively. The sequences were submitted in the NCBI GenBank and accession numbers assigned were MZ519753 and MZ489194. The sequences were analyzed using BLAST search tool in Genbank. The results showed that isolate TAN3PD12 has 99.80% similarity with the sequences of Neurospora sitophila G11 (MN511319.1) (Fig. 6), isolate TAN2PD11 has 96.71% similarity with the sequences of Lichtheimia corymbifera CBS 429.75 (GQ342903.1) (neotype strain) and 97.02% with the sequences of Lichtheimia ornata CBS 291.66 (GQ342946.1) (type strain) (Fig. 6).

Figure 4. Morphology of Neurospora sitophila TAN3PD12 (VCCM 34008) causing tuber rot of cassava. A, B: Colony on Potato Dextrose Agar (PDA). C, D: Sporangiophores and sporangia (X 1000)

Figure 5. Morphology of Lichtheimia sp. TAN2PD11 (VTCC 930027) causing tuber rot of cassava. A: Colony on Potato Dextrose Agar (PDA). B, C, D: Sporangiophores and sporangia (scale bar = 10 µm)

Figure 6. Phylogenetic tree constructed using the Maximum-likelihood method showing the relationships of Lichtheimia sp. TAN2PD11 and Neurospora sitophila TAN3PD12 and their related species constructed based on sequences of the ITS region. Sequences are labeled with their database accession numbers

Re-isolation of the pathogen from symptomatic tuber tissues was performed ac-cording to Koch's postulates. The results showed that the isolated fungal pathogens were the same as the original identified pathogens of cassava that artificially inoculated on to fresh healthy organs of cassava. The established cultures were deposited in the Vietnam Type Culture Collection (VTCC), Institute of Microbiology and Biotechnology, Vietnam National University (VTCC 930027 for TAN2PD11) and Vietnam Culture Collection Microbial (VCCM 34008 for TAN3PD12).

To the best of our knowledge, this is the first report of Lichtheimia sp. and Neurospora sitophila causing tuber rot of cassava. Several studies showed that the fungus Neurospora sitophila causes ripe rot of pear [12]. The tuber rot is the most important cause of loss of cassava production, causing a lot of damage to the farmers, but there are little studies of the fungal pathogens associated with tuber rot of the cassava in Vietnam as well as in the world [1].

The study of the Sankar et al. (2013) showed that, cassava tuber rot accounts for yield losses up to 70% in regions of Kolli hills, Kollam, and Kottayam of South India. Water logging during rain spreads the disease faster. Infected tuber exhibits grey to brown discolouration with watery exudates and foul smell, making it useless for further use. Based on morphological and ITS sequences, the pathogen was identified as Phytophthora palmivora [15]. This results were similar to those found by Arutselvan et al. (2020) which stated that tuber rot of cassava is a serious problem in Tamil  Nadu.  The  disease  is  primarily caused by Phytophthora palmivora, and many secondary invaders also get  associated with the disease at a later stage [3]. In Cameroon 30% of the rotted tubers were infected by Fusarium spp. [4]. Fungal tuber rot have been reported as a limiting factor to cassava production in the humid forests of Central and West Africa. As observed of the authors, the fungi isolated from rotted tubers were quite diverse in identity and there may be differences in the cultivars each pathogen prefers. Pathogens isolated from rot specimens include Armillaria sp., Aspergillus sp., Botryodiplodia theobromae, Fusarium sp., Macrophomina phaseolina, Sclerotium rolfsii and Trichoderma sp. [10]. Rainfall is the most important factor favouring disease infection, soil with poor drainage facilities favours the disease. In addition, rotten tubers left during harvest and ploughed in situ serve as inoculum for the next  crop  and  help  the  pathogen  to  survive  longer  periods  in the soil [3].

This research will be helpful in understanding the pathogen’s behaviour and to combat the disease with efficient management program on the cassava in Vietnam as well as in the world.

Acknowledgments

This study was funded by project ĐTĐL.CN-01/19 (for Pham Viet Cuong) from the Vietnamese Ministry of Science and Technology.

References:

1. Agu KC, Awah NS, Okeke CB, Nwodo MT, Anaukwu CG, Iloanusi CA, Archibong EJ, Ngenegbo U, Mgbemena IC, Umeoduagu N (2016). Mycological Deterioration and Pathogenicity Studies of Post-harvest Cassava. Food Sci. Technol. 4(2): 23-30. DOI:10.13189/fst.2016.040202.
2. Alvarez E, Llano GA and Mejia JF (2012). Chapter 8: Cassava disease. In the book: Ospina Patiño, Bernardo, Ceballos, Hernán. Cassava in the third millennium: modern production, processing, use, and marketing systems. Centro Internacional de Agri-cultura Tropical (CIAT); Latin American and Caribbean Consortium to support Cassava Research and Development (CLAYUCA); Technical Centre for Agricultural and Rural Cooperation (CTA), Cali, CO. 574 (Publicación CIAT no. 377). DOI//hdl.handle.net/10568/54610.
3. Arutselvan R, Pati K, Chauhan VBS and Bansode V (2020). Cassava Tuber Rot, (Phytophthora palmivora) - An Emerging Threat to Cassava Production. Biotica Research Today: 1222-1224.
4. Bandyopadhyay R, Mwangi M, Aigbe SO and Leslie JF (2006). Fusarium species from the cassava root rot complex in West Africa. Phytopathology 96: 673-676. DOI: 10.1094/PHYTO-96-0673.
5. Food and Agriculture Organisation of the United Nations (FAO) Save and Grow: Cassava, A 98 guide to sustainable production and intensification. Rome (2013).
6. Guo H, Li CP, Shi T, Fan CJ, Huang GX (2012). First report of Phytophthora palmivora causing root rot of cassava in China. Plant Disease 96(7): 1072. DOI: 10.1094/PDIS-09-11-0780-PDN.
7. Iti Gontia-Mishra, Niraj Tripathi and Sharad Tiwari (2014). A simple and rapid DNA extraction protocol for filamentous fungi efficient for molecular studies. Indian Journal of Biotechnology, 13, 536-539.
8. Johnson I, Palaniswami A (1999). Phytophthora tuber rot of cassava - A new record in India. Mycol Pl Pathol. 29(3): 323-332.
9. Kibemo B (2017). Isolation, Identifcation, and Characterization of Some Fungal Infectious Agents of Cassava in South West Ethiopia. Adv. in Life Sci. Technol. 54: 16-28.
10. Messiga AJ, Mwangi M, Bandyopadhyay R & Nolte C (2004). The status of fungal tuber rots as a constraint to cassava production in the Pouma district of Cameroon. Proceedings of the 9th Triennial Symposium of the International Society for Tropical Root Crops - Africa Branch, held from 31st October - 5th November 2004, at Whitesands Hotel, Mombasa, Kenya. Yaoundé: IITA, 1-9.
11. Moses E (2007). Guide to Identification and Control of Cassava Diseases. CSIR-Crops Research Institute, Kumasi Ghana.
12. Neurospora sitophila (ripe rot of pear), Hungary. CABI (2019).
13. Ngobisa AICN, Djidjou PK, Godswill NN, Mbenoun M, Simon Z and Dominic F (2015). Isolation and identification of some pathogenic fungi associated with cassava (Manihot esculenta Crantz) root rot disease in Cameroon. African Journal of Agricultural Research: 4539-4542. DOI:10.5897/AJAR2015.10272.
14. Sankar MS, Nath SV, Misra SR & Jeeva LM (2014). Incidence and identification of Cassava tuber rot caused by Phytophthora palmivora. Archives Of Phytopathology And Plant Protection 46(6): 741-746, DOI: 10.1080/03235408.2012.751284.
15. Sankar MSA, Nath VS, Misra RS, Jeeva ML (2012). Inhibitory activity of plant growth regulators on Phytophthora palmivora causing cassava tuber rot. Archives of Phytopathology and Plant Protection. DOI.org/10.1080/03235408.2012.742678.
16. Summerell BA, Gunn LV, Bullock S, Tesoriero LT and Burgess LW (2006). Vascular wilt of basin in Australia. Australasian Plant Pathol. 35: 65-67.
17. White TJ, Bruns T, Lee S, Taylor J (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. San Diego (CA): Academic, 315-322.