Detection, Identification and Phylogenetic Analysis of Sugarcane Pokkah Boeng in Yunnan Sugarcane Areas

Xiaoyan WANG, Xiaoyan CANG, Wenfeng LI, Yinhu LI, Hongli SHAN, Rongyue ZHANG, Yingkun HUANG

Abstract [Objectives] This study was conducted to determine the pathogen species and dominant species of sugarcane pokkah boeng in the sugarcane areas of Yunnan in the low-latitude plateau， so as to provide a basis for the pathogenic mechanism， disease resistance breeding and scientific prevention and control of pokkah boeng.

[Methods]With 14 pokkah boeng samples collected from different sugarcane areas in Yunnan as materials， pokkah boeng-specific detection primers Fv-F4/Fv-R4 and Fp-F3/Fp-R3 were designed based on ribosomal DNA non-transcribed spacer （rDNA-ITS） sequences， respectively， and used to perform PCR on Fusarium verticillioides and F. proliferatum.

[Results] In 12 of the 14 pokkah boeng samples， two species of pokkah boeng pathogens were detected， and there was a phenomenon of composite infection. Seven composite infection samples were selected for sequence alignment and phylogenetic analysis. The sequences of F. verticillioides （GenBank accession number： MZ126549-MZ126555） and F. proliferatum （GenBank accession number： MZ102259-MZ102265） from seven composite infection samples shared 98.6%-100% and 100% with F. verticillioides strain 20 （GenBank accession number： KU508286） and F. proliferatum Dehong strain （GenBank accession number： KJ629482） published in GenBank， respectively. The phylogenetic tree showed that the pathogens of sugarcane pokkah boeng in Yunnan were mainly divided into the F. verticillioides group and F. proliferatum group. In the F. verticillioides group， except for ROC 25 （Lancang， Yunnan） and Funong 10-1405 （Maitreya， Yunnan） which were on an independent branch， the remaining five composite infection samples were grouped with F. verticillioides strains from different geographical origins， and were closely related to F. oxysporum Guangxi strain. All strains of F. proliferatum from different geographic origins were clustered into another group.

[Conclusions]This study provides a scientific basis for disease resistance breeding and disease prevention and control of sugarcane pokkah boeng.

Key words Sugarcane; Pokkah boeng; Dominant species; Phylogenetic analysis

Received： March 5， 2022  Accepted： May 7， 2022

Supported by China Agriculture Research System of MOF and MARA （CARS-170303）; “Yunling Industry and Technology Leading Talent” Training Program "Prevention and Control of Sugarcane Pests" （2018LJRC56）; Special Fund for the Construction of Modern Agricultural Industry Technology System in Yunnan Province （YNGZTX-4-92）.

Xiaoyan WANG （1981-）， female， P. R. China， associate researcher， devoted to research about sugarcane disease.

*Corresponding author. E-mail： huangyk64@163.com.

Pokkah boeng is an important fungal disease of sugarcane worldwide[1-3]. According to reports， the incidence of the disease in India is 51.4%， resulting in a 24.9% reduction in sugarcane production and an annual loss of 25 billion rupees[4-5]. Sugarcane pokkah boeng is distributed in all sugarcane-growing provinces and regions in China. It occurred seriously in Guangxi in the 1980s， and the diseased plant rate of the main cultivar Guitang 11 reached 52.4%[6]. In 1989， Guangdong Yuetang 57-423 and Yuetang 54-176 were highly diseased， which affected local sugarcane production to a certain extent[7]. Since 2000， with the widespread promotion of susceptible varieties such as Xintaitang 1， Xintaitang 10， Xintaitang 16， Xintaitang 22， Xintaitang 25 and Yuetang 93-159 in Chinas sugarcane areas， sugarcane pokkah boeng has been occurring frequently and becoming more serious[3]. Especially in recent years， the rainy and humid climate， coupled with the large-scale planting of susceptible varieties Yuetang 93-159， Xintaitang 25， Xintaitang 1 and Chuantang 79-15， had led to the outbreak of sugarcane pokkah boeng in Lincang， Puer， Yuxi， Honghe and other sugarcane regions in Yunnan， resulting in severe yield and sugar reduction[8]. The survey results showed that the disease was serious in susceptible varieties， often causing the death of a large number of cane stems; the average rate of diseased plants was 81.1%， and it reached 100% in severe cases; the measured yield loss of sugarcane was 38.42% on average and 48.5% in severe cases; and the sugar content in cugarcane decreased by 3.14% on average， and at most 4.21% in severe cases[9].

The pathogens of sugarcane pokkah boeng belong to Fusarium of Deuteromycotina. The disease is caused by Fusarium moniliforme Sheldon in the asexual stage and Gibberella moniliforme Wineland in the sexual stage[10]. There are many species of Fusarium that can cause pokkah boeng， and 7 species have been reported so far， namely F. sacchari， F. verticillioides， F. proliferarum， F. subglutinans， F. andiyazi， F. incarnatum and F. oxysporum[11-13]. The populations and dominant species of pokkah boeng pathogens differ in different countries and sugarcane regions. F. sacchari， F. proliferatum and F. subglutinans occur in Malaysia， and the dominant species is F. sacchari[14]. F. verticillioides， F. proliferatum and F. subglutinans appear in Iran， with F. verticillioides as the dominant species[11-12]. F. sacchari， F. proliferatum and F. andiyazi can be observed in South Africa， and the dominant species is F. sacchari[15]. The species that have been found in the Philippines are F. sacchari， F. proliferatum， F. incarnatum， F. verticillioides and F. subglutinans， of which F. sacchari is the dominant species[13]. F. sacchari， F. verticillioides， F. proliferarum and F. oxysporum have been identified as pathogens that can cause sugarcane pokkah boeng in China， and the dominant species is F. verticillioides[16-18].

Yunnan is the second largest sugarcane planting area in China， which is also the key layout and the most potential development area for Chinas sugarcane production in the future. In recent years， the outbreak of sugarcane pokkah boeng has a clear trend， and susceptible varieties are seriously affected， often causing the death of a large number of sugarcane stems. As a result， sugarcane pokkah boeng， sugarcane pokkah boeng has become the first major disease that causes the most serious reduction in sucrose content and cane yield. Planting and breeding of disease-resistant varieties based on the research of pathogens is the most economical and effective measure to prevent and control pokkah boeng， but there have been few studies on the species， dominant species and phylogenetic evolution among the pathogens of pokkah boeng in Yunnan sugarcane areas， resulting in a lack of reliable evidence for effective prevention and control. In this study， PCR detection of pathogens was carried out on samples collected from different sugarcane areas in Yunnan to identify the main pathogens and dominant species in the region， so as to provide a scientific basis for disease resistance breeding and disease prevention and control of sugarcane pokkah boeng.

Materials and Methods

Experimental materials

The experimental materials were 14 pokkah boeng samples collected in 2020 from 12 sugarcane varieties in Gengma， Menglian， Mengbin， Xinping， Lancang， Shangyun， Kaiyuan and Mile sugarcane regions in Yunnan Province （Table 1）.

PCR detection and identification on dominant species of sugarcane pokkah boeng

Primer design and synthesis

The specific primers of F. verticillioides and F. proliferatum designed based on ribosomal DNA non-transcribed spacer （rDNA-ITS） sequences， namely Fv-F4/Fv-R4 and Fp-F3/Fp-R3， synthesized by Sangon Biotech （Shanghai） Co.， Ltd.， were as follows： Fv-F4： 5′-TCG GGG CCG GCTT GCC GC -3′， and Fv-R4： 5′- TAC AAC TCC CAA ACC CCT GTG AAC ATA C-3′， and Fp-F3： 5′-GTTT TAC TAC TAC GCT ATG GAA GCT-3′， and Fp-R3： 5′-CGA GTT TAC AAC TCC CAA ACC CCT-3′. The expected length of the amplified product was 400 bp.

Extraction of total DNA

First， 0.1 g of leaves was taken， and the total DNA of diseased leaves was extracted using a fungal genomic DNA extraction kit [Sangon Biotech （Shanghai） Co.， Ltd.]. The specific steps were operated according to the instructions， and stored at -20 ℃ for future use.

PCR detection

The PCR amplification system was 25 μl containing 2 μl of DNA template， 12.5 μl of Taq PCR Master Mix， 2.5 μl （10 μmol/L） of upstream and downstream primers， and 5.5 μl of sterilized ddH2O. The PCR reaction program was started with 30 cycles of pre-denaturation at 95 ℃ for 5 min， denaturation at 94 ℃ for 30 s， annealing at 63 ℃ for 15 s and extension at 72 ℃ for 30 s， and completed with a final extension at 72 ℃ for 10 min. The amplified products were detected by 1.5% agarose gel electrophoresis.

Cloning， sequencing and sequence analysis of PCR products

The target fragment was recovered by agarose gel recovery kit [Tiangen Biotech （Beijing） Co.， Ltd.]， and it was ligated and transformed with pEASY-T5 （Beijing TransGen Biotech Co.， Ltd.）. The positive clones were identified by PCR and sent to BGI Genomics. Blast search was performed on the obtained sequences in Genbank， and DNAMAN v6.0 software was used to analyze the nucleotide sequence identity.

Phylogenetic analysis

According to the comparison results， the rDNA-ITS sequences of other sugarcane pokkah boeng pathogens were obtained from the GenBank database. A phylogenetic tree was constructed in the MEGA 6.0[19] software by the Neighbor-Joining method in the Kimura 2-parameter mode with 1 000 bootstrap replicates， using the sugarcane smut Sporisorium scitamineum （GenBank accession number： EF185083） as the outgroup.

Results and Analysis

Molecular identification results of pathogens

With the extracted total DNA of leaves infected with sugarcane pokkah boeng as templates， 14 sugarcane pokkah boeng samples were amplified by PCR with specific primers of F. verticillioides and F. proliferatum， and 12 samples were amplified with a target band 400 bp in size （Fig. 1）， indicating that both F. verticillioides and F. proliferatum were detected. The PCR products of seven composite infection samples were selected for sequencing， and the obtained sequences were submitted to GenBank. The homologous sequences were searched in GenBank with BLAST， and the results showed that the sequences of F. verticillioides （GenBank accession number： MZ126549-MZ126555） and F. proliferatum （GenBank accession number： MZ102259-MZ102265） from seven composite infection samples shared 98.6-100% and 100% with F. verticillioides strain 20 （GenBank accession number： KU508286） and F. proliferatum Dehong strain （GenBank accession number： KJ629482） published in GenBank， respectively. It was further confirmed that the pathogens of sugarcane pokkah boeng in Yunnan sugarcane areas were F. verticillioides and F. proliferatum， and there was a composite infection phenomenon （Table 1）.

Phylogenetic analysis

According to the alignment results， the rDNA-ITS sequences of other sugarcane pokkah boeng pathogens selected and downloaded from the GenBank database were used to establish a phylogenetic tree with the sequences of pokkah boeng pathogens obtained in this study by the Neighbor-Joining method. The phylogenetic tree showed that the pathogens of sugarcane pokkah boeng in Yunnan were mainly divided into the F. verticillioides group and F. proliferatum group. In the F. verticillioides group， except for ROC 25 （Lancang， Yunnan） and Funong 10-1405 （Mile， Yunnan） which were on an independent branch， the remaining 5 composite infection samples were grouped with F. verticillioides strains from different geographical origins， and were closely related to F. oxysporum Guangxi strain. All strains of F. proliferatum from different geographic origins were clustered into another group （Fig. 2）.

Conclusions and Discussion

Sugarcane pokkah boeng is an important fungal disease that seriously affects the high-quality development of Chinas cane sugar industry. The pathogens belong to Fusarium. Although many species of Fusarium can cause pokkah boeng， it is unknown how many of these species cause sugarcane pokkah boeng[16]. In this study， molecular identification methods were applied to detect and sequence 14 sugarcane pokkah boeng pathogens collected from sugarcane areas in Gengma， Menglian， Mengbin， Xinping， Lancang， Shangyun， Kaiyuan and Mile in Yunnan Province， and it was preliminarily confirmed that the pathogens of pokkah boengin the sugarcane areas of Yunnan in the low-latitude plateau were F. verticillioides and F. proliferatum， and there was a common phenomenon of composite infection. These results laid a foundation and provided a basis for the in-depth research and scientific prevention and control of sugarcane pokkah boeng in China.

A phylogenetic tree was constructed on the different sugarcane isolates of Fusarium complex from China. The phylogenetic analysis was performed by neighbor-joining and Kimura 2-parameter method with MEGA 7 software （Kumar et al.， 2016）. Sporisorium scitamineum （EF185083） was used as the outgroup. Numbers on branches indicate bootstrap values based on 1 000 replicates （values<50 are not shown）.

In 2014， Lin et al.[16] conducted pathogen identification and phylogenetic analysis on different sugarcane pokkah boeng pathogens collected from Fujian， Guangdong， Guangxi， Yunnan and Hainan， and determined that the pathogenic Fusarium that caused sugarcane pokkah boeng mainly included gx1 （F. verticillioides） and gx2 （F. proliferatum）. gx1 was the dominant species， accounting for 93% of the total number of strains， and mainly infected sugarcane in summer with high temperature and high humidity， causing pokkah boeng， the damage of which was more serious. gx2 accounted for 7% of the total number of strains， and was relatively resistant to low temperature. It mainly infected sugarcane in winter and caused pokkah boeng damage， but its pathogenicity was weak. Although the pathogens of sugarcane pokkah boeng in Yunnan were also F. verticillioides and F. proliferatum in this study， two pathogens were detected in 12 of the 14 pokkah boeng samples， indicating that the phenomenon of composite infection was common. The coexistence of multiple pathogens in a single plant is an unavoidable fact in the research on the pathogens of pokkah boeng， so it is necessary to further study the mechanism of this coexistence pattern and its role in infection and pathogenesis.

In the phylogenetic analysis， F. verticillioides and F. proliferatum had no obvious variety and geographical differences， but different strains of F. verticillioides from the sugarcane areas of Yunnan in the low-latitude plateau were distributed on different branches， indicating that there is extensive genetic diversity among F. verticillioides strains in Yunnan， and the sugarcane areas of Yunnan in the low-latitude plateau may be a core area of the diversity distribution of F. verticillioides in China.

References

[1] MARTIN JP， HANDOJ OH， WISMER CA. Pokkah boeng. In： Ricaud C， Egan BT， Gi1laspie AG， et al. （eds）， Disease of sugarcane： Major disease[M]. New York： Elsevier Science Pubilishing Co.，Inc.， 1989.

[2] RAID RN， LENTINI RS. Pokkah boeng disease of sugarcane[M]. Florida： Institute of Food and Agricultural Sciences， University of Florida， 2002.

[3] HUANG YK， LI WF， ZHANG RY， et al. Color illustration of diagnosis and control for modern sugarcane diseases， pests， and weeds[M]. Singapore： Springer Nature Singapore Pte Ltd.， 2018.

[4] SINGH OP. Wilt assumes a serious problem in the sugarcane riverine belt of Saraswati Sugar Mills Area， Yamunanagar Ambala[J]. Indian Sugar， 1973（23）： 129-130.

[5] VISWANATHAN R， BALAMURALIKRISHNAN M， KARUPPAIAH R. Yellow leaf disease of sugarcane： Occurrence and impact of infected setts on disease severity and yield[J]. Proceedings of the Sugar Technologists Association of India， 2006（67）： 74-89.

[6] LIU ML， HUANG DF， LI DJ， et al. A preliminary report on the research about the occurrence and control of sugarcane Pokkah Boeng Disease in Guangxi[J]. Guangxi Agricultural Science， 1991（4）： 175-178. （in Chinese）.

[7] HUANG HN， DENG GA， YUAN ZM， et al. The important sugarcane diseases research cooperation group. Survey of the incidence and damage of Pokkah boeng in Dongguan sugar factory area[J]. Sugarcane Canrsugar， 1990（1）： 20-23. （in Chinese）.

[8] LI WF， SHAN HL， HUANG YK， et al. The occurrence epidemic dynamics， control thoughts and countermeasures of important sugarcane diseases in raininess and high humidity season [J]. Sugar Crops China， 2017， 39（2）： 75-77. （in Chinese）.

[9] SHAN HL， LI WF， ZHANG RY， et al. Analysis on epidemic reason of sugarcane pokahh boeng and its losses on yield and sucrose content[J]. Sugar Crops China， 2018， 40（3）： 40-42. （in Chinese）.

[10] SUMAN A， LAL S， SHASANY AK， et al. Molecular assessment of diversity among pathotypes of Colletotrichum falcatum prevalent in sub-tropical Indian sugarcane[J]. World Journal of Microbiology and Biotechnology， 2005， 21（6/7）： 1135-1140.

[11] MOHAMMADI A， NEJAD RF， MOFRAD NN， et al. Fusarium verticillioides from sugarcane， vegetative compatibility groups and pathogenicity[J]. Plant Protection Sciences， 2012， 48（3）： 80-84.

[12] KHANI KT， ALIZADEH A， NEJAD RF， et al. Pathogenicity of Fusarium proliferatum， a new causal agent of pokkahboeng in sugarcane[J]. Proceeding of the International Society of Sugar Cane Technologists， 2013（28）： 1-5.

[13] SAMACO MA， DELACUEVA FM. Molecular characterization of Fusarium spp. Associated with sugarcane pokkah boeng from the Philippines using partial translation elongation factor-1 （TEF-1） gene sequences[J]. Sugar Tech， 2019， 21（4）： 619-630.

[14] SITI NORDAHLIAWATE MS， NUR AIN IZZATI MZ， AZMI AR， et al. Distribution， morphological characterisation and pathogenicity of Fusarium sacchari associated with pokkah boeng disease of sugarcane in Peninsular Malaysia[J]. Pertanika Journal of Tropical Agricultural Sciences， 2008， 31（2）： 279-286.

[15] GOVENDER P， MCFARLANE SA， RUTHERFORD RS. Fusarium species causing pokkah boeng and their effect on Eldanasaccharina Walker （Lepidoptera： Pyralidae）[J]. Proc S Afr Sug Technol Ass， 2010（83）： 267-270.

[16] LIN ZY， XU SQ， QUE YX， et al. Species-specific detection and identification of Fusarium species complex， the causal agent of sugarcane pokkah boeng in China[J]. Plos One， 2014： 9.

[17] BAO YX， SUN YF， LI ZZ， et al. First report of Fusarium oxysporum isolate gx3 causing sugarcane pokkah boeng in Guangxi of China[J]. Plant Disease， 2016， 100（8）： 1785.

[18] MENG JR， HUANG HJ， LI YX， et al. First report of Fusarium sacchari causing sugarcane pokkah boeng in China[J]. Plant Disease， 2020， 104（5）： 1553.

[19] TAMURA K， STECHER G， PETERSON D， et al. MEGA6： Molecular evolutionary genetics analysis version 6.0[J]. Molecular Biology and Evolution， 2013（30）： 2725-2729.