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Sugarcane (Saccharum officinarum) is an economically important crop and is extensively planted across China. In August 2020, leaf midribs with red lesions were observed on cultivar ‘Yunzhe 081609’ in Kaiyuan (103.27°E, 23.71°N), Yunnan, Southwestern China. In July to August 2021, similar symptoms were observed on cultivar ‘Liucheng 05-136’ in Hechi (108.48°E, 24.47°N), Guangxi, and on cultivars ‘Yingyu 91-59’ and ‘Yunzhe 081609’ in Lingcang (99.45°E, 23.33°N), Yunnan. Initially symptoms appeared as red spots on the leaf midribs, which gradually expanded, forming elongated red lesions. At high severity, the leaves broke and hung down. Disease incidence of leaves was estimated at 30 to 50% across the locations. To identify the etiology of this disease, three symptomatic leaves were collected from cultivars ‘Liucheng 05-136’, ‘Yingyu 91-59’, and ‘Yunzhe 081609’, respectively. Symptomatic leaf midribs were cut to small fragments (3 × 5 mm), surface sterilized with 70% ethanol for 30 s followed by 1% NaClO for 1 min, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on potato dextrose agar (PDA), and incubated at 28°C in the dark. Ten isolates with similar morphological characteristics were obtained. Colonies on PDA were white to grayish-white with aerial mycelium growing initially upward and then forming clusters. After 10 days, mycelia turned to grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia became dark brown, septate, longitudinal striate, and measured 21.2 to 25.8 × 11.4 to 16.4 µm (n = 30). Morphologically, the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genomic DNA of four representative isolates (LTGX1, LTGX2, LTYN1 and LTYN2) was extracted using the Ezup Column Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (TEF-1α) gene, and β-tubulin (TUB) gene were amplified with primer pairs ITS1/ITS4 for ITS, EF1-728F/EF1-986R for TEF-1α, and Bt2a/Bt2b for TUB, respectively (Glass and Donaldson 1995; Carbone and Kohn 1999; White et al. 1990), and then sequenced. The ITS (ON533336-ON533339), TEF-1α (ON939550-ON939553) and TUB (OP747306-OP747309) sequences were deposited in GenBank. BLAST searches showed >99% nucleotide identity to the sequences of ex-type isolate CBS 164.96 of L. theobromae (ITS, 99.8% to AY640255; TEF-1α, 99.9% to AY640258; TBU, 100% to EU673110). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodia spp. downloaded from the GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values (100%) to the ex-type isolate CBS 164.96 sequences of L. theobromae. For pathogenicity tests, three healthy 6-month-old potted sugarcane leaf midribs of cultivar ‘Yunzhe 081609’ were wounded with a sterile needle, then inoculated using 8-mm mycelial agar plugs from a 10-day-old culture of strain LTYN1, and covered with wet cotton to maintain high relative humidity. Sterile PDA plugs were used as controls. Plants were placed in a greenhouse at 28 to 32°C. The test was conducted twice. Five days after inoculation, red lesions appeared on the inoculated leaf midribs. These symptoms were similar to those observed in the field. The leaves used for negative controls remained symptomless. The same fungus (L. theobromae) was re-isolated from all inoculated-symptomatic tissues; and isolates had the same morphological traits mentioned above. The DNA sequence data of these isolates was also similar than the original isolates. The association of L. theobromae with S. officinarum was recorded earlier in Cuba (Urtiaga, 1986), Myanmar (Thaung, 2008) and the Philippines (Reinking, 1919). Leaf midribs with red lesions caused by Colletotrichum falcatum has already been described around the world (Costa et al. 2021; Hossain et al. 2021; Xie et al. 2019). All together, this information indicates that L. theobromae is one of the causal agent of the red lesions symptoms on the sugarcane leaf midribs. To our knowledge, this is the first report of L. theobromae causing red lesions on leaf midribs of sugarcane in China. Further research will focus on developing management strategies to control this disease effectively.

Sugarcane white leaf (SCWL) is a phytoplasma disease that has spread rapidly in Yunnan province, China, in recent years. It is now the main barrier to the development of the sugarcane industry in this region. Herein, we explored the insect vector and their dominant population of SCWL phytoplasma at Gengma in Lincang city in 2019, where the occurrence of SCWL is the most serious in Yunnan Province. Two leafhopper species, Tettigoniella viridis (Linnaeus) and Clovia conifer Walker, were tested positive for SCWL phytoplasma by using nested PCR, indicating that they are maybe the potential insect vectors of SCWL phytoplasma. Additionally, nymphs of T. viridis were tested strongly positive, and they were preliminarily identified as the dominant population. Since the main transmission medias of SCWL phytoplasma appears to be SCWL-infected seedcane and the leafhopper insect vectors, establishing a disease-free and healthy seedcane breeding base and eliminating the leafhopper insect vectors in sugarcane planting areas may prevent or control the spread of SCWL and reduce the natural transmission rate in the field. The results expand our theoretical and technical knowledge of sugarcane phytoplasma disease and provide a reference for formulating comprehensive control of SCWL.

Brown stripe is a major fungal disease damaging the leaves of sugarcane. Breeding and cultivating resistant varieties is the most economical and effective method of controlling the disease. The paper reports on the evaluation of sugarcane brown stripe resistance in novel varieties bred by the China Sugarcane System and major varieties under cultivation in the sugarcane cultivating region. Field resistance against brown stripe was identified in 60 novel varieties and in 31 major varieties under cultivation in Lincang, Puer, and Yuxi, in Yunnan Province, and in Yizhou, Guangxi Province, where the incidence and severity of brown stripe is particularly high. Field surveys showed that 32 (53.3%) of the 60 novel varieties were highly resistant to moderately resistant, and 28 (46.7%) were susceptible to highly susceptible. In addition 21 (67.7%) out of the 31 major cultivated varieties were highly resistant to moderately resistant, and 10 (32.3%) were susceptible to highly susceptible. We recommend the intensifying of efforts to eliminate the major varieties that are susceptible, and promotion of the adoption of elite resistant varieties in wet and rainy sugarcane cultivating regions with high incidence and severity of sugarcane brown stripe disease, to effectively manage the brown stripe epidemic.

Sugarcane brown rust, caused by Puccinia melanocephala, is one of the main diseases of sugarcane in China. The identification and discovery of new resistance genes have important theoretical and practical significance for preventing outbreaks of brown rust and ensuring the sustainable production of sugarcane. To screen for polymorphic simple-sequence repeat (SSR) molecular markers for localization of brown rust resistance genes, we used two populations that are suitable for genetic linkage map construction and mapping of new resistance genes to construct resistant and susceptible genetic pools. We then screened 449 pairs of primers to identify polymorphic SSR markers in the parental lines and the resistant/susceptible genetic pools. The results showed that 25 pairs of primers directed amplification of polymorphic DNA fragments between the parents of the cross combination ‘Yuetang 03-393’ × ‘ROC 24’, and 16 pairs of primers amplified polymorphic fragments between the parents of the cross combination ‘Liucheng 03-1137’ × ‘Dezhe 93-88’. Four pairs of primers (SMC236CG, SCESSR0928, SCESSR0636 and SCESSR2551) amplified polymorphic DNA fragments between the parental lines and the resistant/susceptible genetic pools in ‘Yuetang 03-393’ × ‘ROC 24’. The results of this study will establish a solid foundation for the mapping of new brown rust resistance genes, genetic linkage map construction and the development of closely-associated molecular markers in sugarcane.

The aim of this study was to assess brown rust resistance of new sugarcane varieties bred in China and main cultivated varieties in sugarcane planting area, and screen the elite new brown rust resistant varieties for popularization and application in production. In total, 60 new varieties in the national regional test of new sugarcane varieties were tested in Kaiyuan and Lincang, and 34 main cultivated varieties were studied in Lincang, Puer, and Yuxi, Yunnan province, and Yizhou, Guangxi province, China, where the incidence of brown rust was particularly high. The resistance of these sugarcane varieties to brown rust was investigated under natural inoculation and molecular marker-assisted identification was used to detect the brown rust resistance gene Bru1 . The results of field survey showed that 66 (70.21%) of the 94 new and main cultivated varieties were highly resistant to moderately resistant, and 28 (29.79%) were susceptible to highly susceptible. Molecular detection indicated that Bru1 gene was found among 54 (57.45%) of the 94 new and main cultivated varieties. Some main cultivated varieties that were currently planted across large areas such as Guitang 29, Guitang 44, Dezhe 03-83, Liucheng 03-1137, Yuetang 60, and Guitang 46 were highly susceptible to brown rust, and 31 new varieties such as Yuegan 48, Funong 09-2201, Guitang 08-120, Liucheng 09-15, Zhongzhe 1, Yunzhe 08-1609, Yunrui 10-187, and Zhongtang 1201 were resistant. Therefore, in the sugarcane planting areas with high incidence of brown rust and wet and rainy climates, more effort should be eliminated the main susceptible varieties and promoted the application of new resistant varieties. This will help to achieve a reasonable distribution of varieties, fundamentally control the outbreak of disease in sugarcane planting areas, and provide security for the high-quality development of sugarcane industry in China in the future.

Pokkah boeng is an epidemic fungal disease that greatly affects the development of the sugarcane industry in China. Resistance to pokkah boeng disease differs among sugarcane varieties. Breeding and planting resistant cultivars is the most economic and effective measure for controlling this disease. In this study, new sugarcane varieties bred by sugarcane research units in China and the major varieties cultivated in various sugarcane areas, were screened to find new elite pokkah boeng-resistant varieties for application in sugarcane production. Sixty new varieties resistance to pokkah boeng disease were evaluated in regionalized experiments under field conditions. Meanwhile, the resistance of the 31 main cultivated varieties was analyzed under field conditions in Lincang, Puer, and Yuxi, Yunnan Province, and in Yizhou, Guangxi Province, where the incidence of pokkah boeng is particularly high. Field surveys showed that 35 (58.3%) of the 60 new sugarcane varieties were highly resistant to moderately resistant, and 25 (41.7%) were susceptible to highly susceptible. Of the 31 main cultivated varieties, 15 (48.4%) were highly resistant to moderately resistant, and 16 (51.6%) were susceptible to highly susceptible. The results suggested that, in wetter and rainy sugarcane cultivation areas with high incidence of pokkah boeng disease, it is necessary to increase efforts to eliminate the susceptible major cultivated varieties and promote the cultivation of novel resistant varieties. This will help to achieve a reasonable distribution of varieties, fundamentally control the outbreak of pokkah boeng disease, and facilitate the high-quality development of the sugarcane industry in China.

The aim of this study was to assess brown rust resistance in 60 new sugarcane varieties bred by the China Sugarcane System in recent years and in 34 major varieties cultivated in sugarcane growing areas in China. The resistance of these sugarcane varieties to brown rust was investigated in the field, and molecular markers were used to detect the brown rust-resistance gene Bru1. The results of the field survey showed that 66 (70.21%) of the 94 sugarcane varieties were highly resistant to moderately resistant, and 28 (29.79%) were susceptible to highly susceptible. The Bru1 gene was detected in 54 (57.45%) of the 94 sugarcane varieties. Seven highly resistant varieties and five resistant varieties did not carry Bru1, suggesting that they carry other genes associated with brown rust resistance. This study provides a scientific basis and identifies disease-resistant germplasm for selection of plants for sugarcane production.

Sugarcane brown rust induced by Puccinia melanocephala is an important global disease. Exploring novel resistance genes and breeding varieties with durable resistance is the most economical and effective way of controlling brown rust. The establishment of complete and high-density molecular genetics maps is a key prerequisite for the discovery and location of sugarcane brown rust resistance genes. To obtain more polymorphic simple sequence repeat (SSR) markers for the construction of genetic linkage maps, six highly resistant varieties and six highly susceptible varieties were used as parents to screen the primers with clear bands, obvious polymorphism, and good repeatability. The highest numbers of polymorphic primers were obtained from the parental group of Liucheng 03-1137 × Dezhe 93-88 (52.38%). Ten pairs of primers including mSSCIR34 exhibited high polymorphism in the parental group of Liucheng 03-1137 × Dezhe 93-88. Here, we recommend ten SSR pairs of polymorphic primers including mSSCIR34 to use in the mapping populations of Liucheng 03-1137 × Dezhe 93-88. The polymorphic primers could facilitate the construction of genetic maps and lay an appropriate foundation for the localization of brown rust resistance genes and development of molecular genetics linkage markers.

We assessed inheritance of resistance to sugarcane brown rust (Puccinia melanocephala) in selfing F1 populations of wild sugarcane germplasm Erianthus rockii ‘Yundian 95-19’ and E. rockii ‘Yundian 95-20’. We tested parent and selfing F1 individuals for the brown rust resistance gene, Bru1, that has been shown to confer resistance to brown rust in sugarcane. The Bru1 gene was not detected in E. rockii ‘Yundian 95-19’, E. rockii ‘Yundian 95-20’ or their selfing F1 individuals, and we found there was segregation of resistance in the two selfing F1 populations (segregation ratio: 3:1). The results confirmed resistance in E. rockii ‘Yundian 95-19’ and E. rockii ‘Yundian 95-20’ to sugarcane brown rust is controlled by a novel, single dominant gene.

Sugarcane leaf scald caused by Xanthomonas albilineans (Ashby) Dowson is an extremely destructive disease. In 2017, suspected symptoms of sugarcane leaf scald was observed, in Baoshan, Mengding and Jinping, Yunnan Province, China. The bacteria was isolated from the symptomatic stalks and was subsequently identified as the causal agent using Koch’s postulates. BLASTN analysis showed that the amplified nucleotide sequences of 16S rRNA, gyrB and rpoD genes from all isolates shared 100% sequence identity with the nucleotide sequences of corresponding genes of X. albilineans strain GPE PC73 (Acc no. FP565176). Based on symptom diagnosis in the field, colony morphology, validation of Koch’s postulates and molecular identification of 16S rRNA, gyrB and rpoD genes, the disease was confirmed as sugarcane leaf scald caused by X. albilineans. Partial 16S rRNA, gyrB and rpoD gene sequence analysis of 36 X. albilineans strains from Yunnan and Guangxi provinces, China, was determined. The results revealed that the gyrB gene was the most sensitive in differentiating X. albilineans strains from Yunnan and Guangxi provinces.

Ratoon stunting disease (RSD) and brown rust are major plant diseases that disrupt sugarcane production. Identifying disease-resistant parental lines offers an efficient strategy for controlling these diseases in sugarcane. The aim of this study was to clarify the occurrence of RSD and the distribution of the major brown rust resistance gene Bru1 among commonly used sugarcane breeding parents in China. We used PCR assays for the molecular detection of RSD and Bru1 in 255 sugarcane breeding parents collected from the National Germplasm Repository of Sugarcane of China (2019–2020). RSD was detected in 68.6% of the parental lines, indicating that commonly used sugarcane breeding parents in China are severely infected by RSD. However, 80 of the tested lines were RSD negative, indicating these parental lines had good resistance to RSD. The RSD incidence rate varied between different parental line series: VMC had the lowest RSD frequency (40.0%) and Guitang had the highest frequency (84.6%), indicating that the VMC series had good resistance levels to RSD and that a high proportion of parental lines were RSD resistant. Bru1 was present in 87 of the 255 tested parental lines, with greatest frequency detected in the ROC series (71.4%) and lowest frequency detected in the CP series (21.6%). These results indicate that Bru1 is a major source of brown rust resistance in commonly used sugarcane breeding parents in China, with important implications for brown rust resistance breeding programs in sugarcane. Overall, these results provide a reference for selecting appropriate breeding lines that are resistant to RSD and brown rust, offering guidance for effective disease control in sugarcane.

Sugarcane (Saccharum officinarum L.) is the main sugar crop in China. Yunnan is the second largest sugarcane production province in China. In December 2018, leaf blight was first observed on almost every leaf of sugarcane on 'Huanan 54-11', 'Baimei' and 'Chongan' in Kaiyuan (103°27' E, 23°72' N), Yunnan. In October 2019, during our survey in the field in Lingcang (100°08' E, 23°88' N), Yunnan, this disease was also observed on 'ROC 25'. Symptoms of the disease initially appeared as wilted, which seemed to be cause by water stress. As the disease progressed, irregular straw-yellow and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath, many small black conidia formed in the dead leaf tissue under humid conditions. Symptomatic leaf tissues were surface-sterilized with 70% ethanol for 30 s, 0.1% HgCl2 for 1 min, and rinsed with sterilized water three times, air dried on sterile filter paper, and plated on potato dextrose agar (PDA). Six isolates were obtained from six symptomatic leaf samples and were transferred onto potato carrot agar (PCA). Colonies on PDA were white with loose aerial hyphae at first, then turned to dark olive or dark. Colonies on PCA were grayish with sparse hyphae, then turned to dark gray. Conidiophores were brown, simple or branched, and produced numerous conidia in short chains. Conidia (n = 50) were obclavate to obpyriform or ellipsoid, brown to dark brown, with a cylindrical short beak at the tip (2.3 to 17.3 µm in length), and 15.3 to 46.6 μm × 4.2 to 17.9 μm, 2 to 7 transverse septa and 0 to 3 longitudinal septa. Morphologically, the isolates were identified as Alternaria tenuissima (Simmons 2007). Two representative isolates C4 and C5 were selected for molecular identification. The internal transcribed spacers (ITS), Histone 3 genes and plasma membrane ATPase were amplified with primer pairs ITS1/ITS4, H3-1a/H3-1b and ATPDF1/ATPDR1, respectively (Glass et al. 1995; Lawrence et al. 2013). The sequences were deposited in GenBank (ITS, MT679707-MT679708; Histone 3, MT710929-MT710930; ATPase, MT833928-MT833929). BLAST searches showed ≥99% nucleotide identity to the sequence of A. tenuissima (ITS, 100% to MN822571; Histone 3, 100% to MN481955; ATPase, 99% to JQ671875, 100% to MH492703, respectively). Thus, the fungus was identified as A. tenuissima based on morphological and molecular characteristics. For pathogenicity tests, five healthy 2-month-old potted sugarcane leaves were wounded with one sterile needle and inoculated with 20 μl of suspension of 106 conidia/ mL, and five plants were inoculated with distilled water as the controls. Plants were placed in a greenhouse at 25 to 35°C. After two months, the leaf wound inoculated with the putative pathogen displayed blighted as those observed in the field whereas the controls remained symptomless. The fungus was reisolated from symptomatic leaves with the same morphological and molecular traits as the original isolates. The fungus was not isolated from the control plants. Pathogenicity tests were repeated two times. A. tenuissima causing leaf blight on barley in China was reported in 2008 (Luo et al. 2008). Leaf spot disease of sugarcane caused by A. tenuis has been recorded in Maharashtra (Patil et al. 1974). To our knowledge, this is the first report on A. tenuissima affecting leaf blight on sugarcane in Yunnan Province, China. Identification of the causes of the disease is important to develop effective disease management strategies. The author(s) declare no conflict of interest. Funding: This research was supported by Sugar Crop Research System (CARS-170303), the Yunling Industry and Technology Leading Talent Training Program "Prevention and Control of Sugarcane Pests" (2018LJRC56), and the Yunnan Province Agriculture Research System. References: Glass, N. L., et al. 1995. Appl. Environ. Microbiol. 61:1323. Lawrence, D. P., et al. 2013. Mycologia 105:530. Luo, Z., et al. 2008. Acta Phytophy. Sin. 35(5): 469-470. Patil, A.O., et al. 1974. Res. J. Mahatma Phule Agric. Univ. 5(2): 122-123. Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands. Caption for supplementary Figure 1 Supplementary Figure S1. Disease symptoms of sugarcane leaf blight disease and morphological characteristics of Alternaria tenuissima. (A) Typical straw-yellow and blighted lesions on naturally-infected leaves of sugarcane; (B) Infected symptoms on wounded leaves of sugarcane two months after artificial infection with A. tenuissima; (C) Colony of A. tenuissima on PDA; (D) Colony of A. tenuissima on PCA; and (E-F) Sporulation and conidia of A. tenuissima on PCA. (Scale bars = 100 μm; 20 μm).

Sugarcane white leaf (SCWL) is a devastating sugarcane (Saccharum officinarum) disease caused by a 16SrXI group phytoplasma, which is extremely harmful to sugarcane production. To determine the occurrence of SCWL in different varieties in 2018, we conducted a field survey and performed nested PCR detection of SCWL phytoplasma in cane-planting areas of Mangweng and Hepai in Gengma, Yunnan province, which are the areas most severely affected by SCWL in China. The results of the field survey showed that the symptomatic incidence of SCWL differed among varieties. The mean symptomatic incidence of SCWL on variety Yuetang60 was the highest (73.50%), and it was the lowest on Liucheng05-136 (13.67%). Using nested PCR, the SCWL phytoplasma was detected in symptomatic plants of all varieties more than 90% of the time; the SCWL phytoplasma was detected in 91 and 97% of symptomatic plants of Yingyu91-59 and Liucheng05-136 varieties, respectively. The SCWL phytoplasma was detected by PCR in 82% of the asymptomatic plant samples. The results of this study showed that field survey based on white leaf symptoms did not accurately reflect the actual occurrence of the SCWL phytoplasma.

Sugarcane mosaic disease is one of the most serious and prevalent viral diseases of sugarcane in China. Resistant varieties are the most economical and effective measures for controlling this disease. Resistance against Sugarcane streak mosaic virus (SCSMV) and Sorghum mosaic virus (SrMV) in 41 excellent sugarcane innovation germplasms and their parents (Saccharum officinarum L. “Ludashi” × Erianthus rockii Keng “Yundian 95-19”) was checked by using a stalk inoculation and RT-PCR detection during 2015 and 2016. Results indicated that among the 41 excellent sugarcane innovation germplasms and parents, 23 were highly (Grade 1) to moderately resistant (Grade 3) to SCSMV, meanwhile, 31 were highly (Grade 1) to moderately resistant (Grade 3) to SrMV. Ten germplasms were highly resistant (Grade 1) to resistant (Grade 2) to both SCSMV and SrMV. Among these, six germplasms (Yun 09-604, Yun 09-607, Yun 09-619, Yun 09-633, Yun 09-656, Yundian 95-19) were highly resistant (Grade 1) to both SCSMV and SrMV, accounting for 13.95% of the total germplasm materials. These results may provide resistance resource available for the breeding program of sugarcane cultivars against the major vrius associated with mosaic disease in China.

Field resistance against sugarcane thrips (Fulmekiola serratus) in 21 sugarcane varieties was tested following inoculation with a natural population. The results revealed differences in pest incidence levels in different varieties and between different leaf positions in the same variety. The tested varieties were subsequently categorized into four levels of resistance (Grades 1–4). The variety GT31 and LC05-136 showed the highest resistance (Grade 1), with a pest index of less than 0.35, while 12 varieties including FN91-21 and FN39 had a pest index of 0.35–0.45 (Grade 2). Five varieties including YT60 and YZ03-194 had a pest index of 0.45–0.55 (Grade 3), while FN38 and DZ03-83 had the lowest resistance (Grade 4), with a pest index of greater than 0.65. Correlation analysis further revealed that the number of thrips present on a single plant was related to the level of resistance; however, the correlation was relatively weak. These findings provide a preliminary evaluation of thrips resistance screening in sugarcane, laying the foundation for analysis of thrips-resistant genes, breeding of thrips-resistant varieties and resistance evaluation in new sugarcane germplasms.

Sugarcane streak mosaic virus (SCSMV) is one of the most important pathogens associated with sugarcane mosaic disease. In recent years, the rapid spread of SCSMV in Yunnan province, China, has raised concerns. In this study, we analyzed 110 sugarcane samples with mosaic symptoms taken from main growing areas in Yunnan province. RT-PCR detection results showed that 94% samples were positive for SCSMV. We subsequently analyzed the genetic diversity and population structure of SCSMV based on the coat protein (CP) sequences of 48 isolates. The overall mean value of nucleotide diversity for all isolates was 0.01127 ± 0.00082. Phylogenetic analysis based on CP gene sequences showed that all the Chinese isolates were clustered into one group, showing a distinct geographic feature. Analysis of population structure showed that the population from this study is not genetically different from that in China, but is genetically different from populations from other countries. Our results suggest that SCSMV has become the main pathogen of sugarcane mosaic disease in Yunnan province, China and that the recent rapid spread of SCSMV in Yunnan is not due to the invasion of new SCSMV populations.

Sugarcane mosaic disease is one of the most serious and prevalent viral diseases of sugarcane in China. The use of resistant varieties is the most economical and effective measures for controlling this disease. Sugarcane streak mosaic virus (SCSMV) and Sorghum mosaic virus (SrMV) are the two predominant pathogens of mosaic disease in the cane-growing regions of China. In 2015, 2016, double resistance to SCSMV and SrMV was identified once a year using a combination of artificial stalk-cutting inoculation and RT-PCR detection in 71 new elite sugarcane varieties/clones. Of the 71 new elite sugarcane varieties/clones, 24 were highly (Grade 1) to moderately resistant (Grade 3), and 47 were susceptible (Grade 4) to highly susceptible (Grade 5) to SCSMV. Furthermore, 27 were highly (Grade 1) to moderately resistant (Grade 3), and 44 were susceptible (Grade 4) to highly susceptible (Grade 5) to SrMV. Fifteen new elite sugarcane varieties/clones were highly resistant (Grade 1) to resistant (Grade 2) to SCSMV and SrMV. While, five new elite sugarcane varieties/clones (Yuegan 34, Yuetang 55, Yunzhe 03–258, Yunzhe 05–51, Yunzhe 06–80) were highly resistant (Grade 1) to SCSMV and SrMV. We defined the resistance of 71 new elite sugarcane varieties/clones to the two main pathogens of mosaic disease and identified 15 resistant to both SCSMV and SrMV. These results provide an elite resistance resource for effective prevention and control of mosaic disease and may serve as a reference for commercial varieties.

Brown rust, caused by Puccinia melanocephala has become a serious problem for sugarcane production in China. Therefore, 50 new elite varieties bred by the Chinese Sugarcane System were screened for brown rust resistance and the presence of a major resistance gene, Bru 1. Identification of field resistance and molecular detection of the brown rust resistance gene Bru 1 in the 50 new elite varieties and 2 main cultivated varieties were carried out in 2014 and 2015 at regional experimental stations of the China Sugarcane System in Dehong and Baoshan, Yunnan Province, where the incidence of brown rust is particularly high. Resistance evaluations based on ratings of symptom severity resulting from field infection showed that 32 (64%) of the 50 new elite varieties were highly resistant (n = 13, 26%), resistant (n = 16, 32%) and moderately resistant (n = 3, 6%). Molecular detection identified Bru 1 in 27 (54%) of these resistant varieties suggesting that brown rust resistance in these new elite varieties is primarily controlled by Bru 1. The absence of Bru 1 in the remaining five resistant suggested the presence of additional brown rust resistance genes. Two main cultivated varieties were highly resistant and Bru 1 - positive varieties. Different series differed in the frequencies of resistant and Bru 1 - positive varieties. Yunzhe series varieties had the highest frequency of resistant varieties (87.5%) and the highest frequency of Bru 1-positive varieties (81.3%); In contrast, the Yuetang series varieties had the lowest frequency of resistant varieties (40%) and the lowest frequency of Bru 1-positive varieties (30%). In this study, we defined the field resistance of 50 new elite varieties to brown rust, and determined the distribution of brown rust resistant gene Bru 1 in different series varieties. These results will provide new options for commercial production with effective resistance to brown rust. Moreover, these new elite resistant varieties can be used for subsequent breeding and selection to produce agronomically superior varieties with effective brown rust resistance.

To confirm the phytoplasma association in 13 sugarcane white leaf (SCWL) symptomatic samples collected from Puer, China, nested PCR assays with phytoplasma universal primer pairs was performed. A ~ 1240 bp product was obtained from 10 out of 13 symptomatic samples, and 16S rRNA gene sequencing results showed that all sequences of SCWL isolates were 100% identical to each other. BLASTn analysis revealed 100% sequence similarity with the 16S rRNA genes from SCWL phytoplasma strains LC7 and LC9 from Lincang, Yunnan province, China belonging to 16SrXI-B subgroup. Virtual restriction fragment length polymorphism also confirmed that Puer SCWL strains were member of the 16SrXI-B subgroup.

In 2015, symptoms of red stripe were first viewed on leaves of sugarcane plants growing in Yuanjiang, Yunnan Province. Symptomatic leaf samples were collected from infected plants and isolations were made on NA medium. Pure culture isolates from resulting single colony were identified based on cultural and morphological characteristics as Acidovorax avenae subsp. avenae. Morphological identification was confirmed by sequence analysis of 16S rDNA. Pathogenicity tests confirmed Yunnan isolates of Acidovorax avenae subsp. avenae as the causal agent of red stripe on sugarcane. This is the first report of this disease in Yuanjiang, Yunnan, China. Field surveys showed that the sugarcane cultivar ‘Yunzhe03-194’ was highly susceptible, and disease incidence varied from 8 to 80%. Nine other sugarcane cultivars were found to be symptomless in the field. Further research is required to confirm the resistance levels of the sugarcane varieties.

Seventy-seven mosaic leaf samples from new sugarcane varieties from national regional trials in Kaiyuan, Mile and Yuanjiang, Yunnan Province, China, were collected in 2015 and analyzed using reverse-transcription polymerase chain reaction (RT-PCR) to determine the pathogen causing mosaic symptoms in sugarcane. Three viruses, Sugarcane streak mosaic virus (SCSMV), Sorghum mosaic virus (SrMV) and Sugarcane mosaic virus (SCMV), were detected in 100, 27.3 and 1.3% of the samples respectively. Co-infection with SCSMV, SrMV and SCMV was also found for the first time in the new variety Qiantang 5. Nucleotide sequences of the coat protein (CP) gene of 48 SCSMV and 17 SrMV isolates were subsequently sequenced and analyzed with the CP gene sequences of SCSMV and SrMV reported in GenBank to perform phylogenetic analyses. Geographic differences were observed in the phylogenetic tree of SCSMV and all SCSMV isolates from China clustered into a single China group. Moreover, SCSMV isolates from different new varieties were distributed across all subgroups or clustered into an independent branch in the China group. SrMV isolates obtained in this study and those published in GenBank clustered into two groups and were distributed into different subgroups within these groups. Furthermore, the distribution of SrMV isolates from different new varieties overlapped in these different groups and subgroups, with no obvious geographic differences. These results provide a scientific basis for sugarcane mosaic or streak mosaic disease resistance breeding and effective control of this disease.

The mixed-mode chromatography supports were prepared, including anion exchange and hydrophobic interaction chromatography. The matrix was based on the gigaporous polymer microspheres from copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. The microspheres were modified by poly(ethylene imine) and butyl glycidyl ether for mix-mode chromatography. The effects of ion capacity and hydrophobic ligand density on protein capacity and recovery were evaluated. The results indicated that the protein capacity and recovery increased with ion capacity of the supports in the range of 0.2–0.5 M. The max capacity was 40 mg mL−1 and the protein recovery was more than 90%. The hydrophobic interaction chromatography occurred when hydrophobic ligand density was more than 0.03 M. The backpressure on these supports was less than 2 MPa at flow rate > 2000 cm h−1. In addition, the high resolution could be retained even at high flow rate of 2880 cm h−1 for purification of Ig G from human serum. Therefore, this support showed a large potential in high through-put separation.

Brown rust caused by Puccinia melanocephala H. Sydow & P. Sydo is an important sugarcane disease that severely threatens sugarcane production in China. Breeding and planting resistant cultivars is the most cost-effective and efficient strategy for the control the disease. In addition, exploration, research, and utilization of resistant genes are the basis for resistance breeding. In the present study, four sugarcane varieties without the Bru1 gene and highly resistant to brown rust were used as male parents, and four varieties highly susceptible to brown rust were used as the female parents to configure hybrids. Six F1 hybrid populations were subjected to authenticity determination using SSR markers, brown rust resistance phenotype identification using artificial inoculation, and molecular detection of Bru1 gene. The results showed that the genetic segregation ratios in F1 hybrid populations of Yuetang 03-393 (HS) × ROC24 (HR) and Liucheng 03-1137 (HS) × Dezhe 93-88 (HR) were 3R:1S and 1R:3S, respectively, and Bru1 gene was not detected in their F1 hybrids. The results indicated that two dominant novel genes controlled brown rust resistance in Yuetang 03-393 (HS) × ROC24 (HR) and two recessive novel genes controlled brown rust resistance in Liucheng 03-1137 (HS) × Dezhe 93-88 (HR). Two genetically segregating populations that could be used for the localization of novel brown rust resistance genes were developed in the present study, and could lay a suitable foundation and provide theoretical reference for the analysis of the genetic mechanism of brown rust resistance, genetic map construction, mapping of novel resistance genes and the development of closely associated molecular markers in the future.

Sugarcane brown rust induced by Puccinia melanocephala is an important global disease. Bru1 is a major brown rust resistance gene and has been shown to provide broad-spectrum resistance against various brown rust pathogen strains originating from different countries. Thus, brown rust resistance genes have important implications for sugarcane brown rust resistance breeding programs. The objectives of this study were to (1) assess the level of resistance of common sugarcane crossbreeding parents to P. melanocephala, (2) determine the distribution of the Bru1 gene in common sugarcane crossbreeding parents in China, (3) screen, evaluate, and select rust-resistant parental lines to improve the efficiency of breeding for brown rust resistance. Brown rust resistance in 101 common sugarcane breeding parents was collected from the Chinese National Nursery of Sugarcane Germplasm Resources and was scored and identified at the seedling stage and was assayed for the presence of the brown rust resistance gene Bru1. The results showed that Bru1 was detected in 48 (47.5 %) resistant clones out of the 101 tested parental lines, indicating that brown rust resistance in common sugarcane breeding parents in China is primarily controlled by Bru1. The apparent absence of Bru1 from the other 29 resistant and 24 susceptible parental lines suggested that they may carry brown rust resistance-associated genes other than Bru1. These results may facilitate future efforts to breed varieties with improved resistance to brown rust and may provide additional resistant parental lines for selecting and planting elite-resistant varieties to effectively control brown rust disease in sugarcane.

Sugar cane white leaf (SCWL) is a serious disease caused by phytoplasmas. In this study, we performed nested PCR with phytoplasma universal primer pairs (P1/P7 and R16F2n/R16R2) for the 16S rRNA gene to detect SCWL phytoplasmas in 31 SCWL samples collected from Baoshan and Lincang, Yunnan, China. We cloned and sequenced the nested PCR products, revealing that the 16S rRNA gene sequences from 31 SCWL samples were all 1247 bp in length and shared more than 99 % nucleotide sequence similarity with the 16S rRNA gene sequences of SCWL phytoplasmas from various countries. Based on the reported 16S rRNA gene sequence data from SCWL isolates of various countries, we conducted phylogenetic and virtual RFLP analysis. In the resulting phylogenetic tree, all SCWL isolates clustered into two branches, with the Lincang and Baoshan SCWL phytoplasma isolates belonging to different branches. The virtual RFLP patterns show that phytoplasmas of the Lincang branch belong to subgroup 16SrXI-B. However, the virtual RFLP patterns revealed by HaeIII digestion of phytoplasmas of the Baoshan branch differed from those of subgroup 16SrXI-B. According to the results of phylogenetic and virtual RFLP analysis, we propose that the phytoplasmas of the Baoshan branch represent a new subgroup, 16SrXI-D. These findings suggest that SCWL is caused by phytoplasmas from group 16SrXI, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D.

For many years, the control and treatment of sugarcane pests in China mainly relies on highly toxic and broad-spectrum chemical pesticides, such as methamidophos and omethoate. As a result, many nontargets, natural enemy species rapid declines, the dynamic relationship between pests and their natural enemies were disrupted, and pests develop resistance, natural enemy population decreased. It alleviates the function on natural factor of controlling pests and makes the prevention and control work more passive. Moreover, the misuse of pesticides leads to pesticide residue, environment pollution, and human health harmfulness. Therefore, a new task for plant protection workers is to control the pests in a comprehensive and environmentally friendly way. In recent years, biological control has become one of the important measures in integrated pest control, and its social and ecological benefits are attracting more and more attention from the state and society (Pu 1984; Bao 1988; Pu and Li 1996; Zhou et al. 1999). The study and development of using dominant natural enemies to control the pests can get the obvious effect on increasing yield, reducing environmental pollution, maintaining ecological balance, saving energy, and cutting down production cost (Yang et al. 2006; An et al. 2009). With China’s accession to the WTO, it is becoming increasingly important to protect and utilize the natural enemies of sugarcane pests, when the implementation of the process of quality standardization of agricultural products has been continuously promoted.

There are more than 120 sugarcane diseases that have been found in the world presently (Chen 1982; Rott et al. 2000), and more than 60 have been reported in China (The important sugarcane diseases research cooperation group 1991; Lu et al. 1997; Huang and Li 2014, 2016). Mastering the disease types, damage, and distribution in the sugarcane area can provide scientific basis for the sugarcane disease-resistance breeding, quarantine of introduced varieties, and disease management and research. Sugarcane production areas in China have drought in winter and spring and hot climate and good rainfall in summer and autumn. With the rise of temperature in spring, in case of cold current as well as the rainfall and temperature decline and the poor management, planted sugarcane in winter and this season are easily infected by pineapple disease and the ratoon crops are easily damaged by red rot disease. Because the whole seedling stage is in the dry season, smut becomes substantial causing sparse breakup. After June, with the high temperature and humidity, eyespot, brown stripe, yellow spot, pokkah boeng, rust, ring spot, and leaf scorch diseases are easily occurring and epidemic. Especially in recent years, introducing seedcanes frequently and transposing seedcanes in sugarcane planting area make some dangerous seedcane-transmitted diseases (ratoon stunting disease, mosaic, smut, yellow leaf syndrome, etc.) which are spread by seedcane between sugarcane areas. Then, caused sugarcane diseases occur commonly and damage more severely; have a serious impact on sugarcane yield, quality, and ratooning capacity; and bring serious hidden trouble to the safe production of sugarcane. In order to scientifically control the spread and damage of sugarcane disease, enhancing the capabilities of disaster prevention and reduction and ensuring the quality of sugarcane variety and safety of sugarcane production in 20 diseases that commonly occur in sugarcane production are systematically described in this chapter with clear-colored photos and scientific, accurate words. The content includes the occurrence and damage, identification of symptoms, characteristics of epidemic infection, and control measures.

Using chemical herbicides to replace artificial and mechanical weeding in the field is a widely used technology worldwide. It represents low-cost prevention and control, saves working hours, reduces labor intensity, and can effectively prevent weeds in a timely manner, thus ensuring normal growth of sugarcane plants for high yield (Su and Song 1996). Improper use of chemical herbicides can cause phytotoxicity, which leads to abnormal sugarcane normal growth and yield reductions. The correct use of herbicides, based on the weed species and scientific spraying according to the soil moisture content, can be effective to control weeds in sugarcane fields. This chapter uses clear color photos and scientific, accurate text to describe systematically the main weeds of sugarcane fields and their distribution, growth and decline law in the field, and chemical weed control measure.

Sugarcane white leaf (SCWL) phytoplasma was identified from sugarcane varieties in fields of Lincang and Baoshan, and imported sugarcane varieties from Myanmar, Philippines and Thailand in quarantine glasshouse at Yunnan Province, China. The associated phytoplasmas were characterized as strains of 16SrXI group by using MLOX/MLOY and P1/P2 primer pairs, 16Sr sequence identity and phylogenetic relationships.

Sugarcane white leaf (SCWL) is an important disease caused by a phytoplasma. In Baoshan, Yunnan, China, SCWL was firstly observed in 2012, and has extended its area of occurrence to 600 hm² . Up to 52% of the plants may become diseased in a field and even complete loss of cane yield may result in the heavily infected fields, posing a serious threat to Yunnan sugar industry. To ascertain the causal agent of suspected SCWL disease in Yunnan, nested PCR using two sets of phytoplasma primer pairs (MOLX/MLOY and P1/ P2) was used to successfully amplify a genomic region of the 16S ribosomal DNA (16S rDNA) from 36 suspected SCWL samples. On the basis of sequencing, phylogenetic analysis and nucleotide alignments of 17 nested PCR products randomly selected from positive samples, identical fragments of 210bp in length were obtained that could be clustered into the 6Sr group XI (Rice Yellow Dwarf group) and shared 100% identity with the 16S-23S intergenic spacer region (ISR) of a member of this group, the SCWL phytoplasma (GenBank: HQ917068), and 99.52% with Sugarcane grassy shoot phytoplasma, of the same group. These results indicate that the SCWL disease in Baoshan is caused by a phytoplasma of the 6Sr group XI.

Exploring and utilizing resistant germplasm resources plays a pivotal role in breeding for disease resistance, while screening resistant germplasm is important for selecting and breeding varieties resistant to disease. In the present study, we used PCR to detect the ratoon stunting disease (RSD) bacterium Leifsonia xyli subsp. xyli (Lxx) in 137 sugarcane core germplasms from the National Nursery of Sugarcane Germplasm Resources (NNSGR, Kaiyuan, China) in 2009, 2010 and 2011. A total of 21 germplasms that tested negative for Lxx in 2009 and 2010 were selected for further Lxx detection after being subjected to artificial inoculation in 2011 and 2012. The 21 core germplasms that were negative for Lxx after natural infection and artificial inoculation can provide elite resistance source materials and reference frames for the effective breeding of RSD-resistant sugarcane varieties. Under natural conditions, 116 (84.67%) and 21 (15.33%) out of 137 germplasms were positive and negative for Lxx, respectively, as determined by PCR detection, which suggests that a relatively high ratio of sugarcane core germplasms was sensitive to RSD, while few were resistant to RSD. The sequencing and analysis of 30 randomly selected PCR products showed that all 30 sequences were identical or highly homologous to the corresponding Lxx genome region published in GenBank (99.54–100% similarity). Lxx can be detected effectively and precisely by PCR. We therefore recommend PCR as a rapid, low cost and simple procedure to score sugarcane core germplasms for RSD resistance.

The introduction of the sugarcane white leaf (SCWL) phytoplasma into Yunnan Province, China was confirmed for the first time by nested PCR. Sequencing and analysis of 17 nested PCR products of the 16S rDNA of the phytoplasma from random samples showed that these sequences were identical. A BLAST search indicated that the sequences were highly homologous (99.05–100% similarity) to the corresponding genome region in GenBank. Field surveys showed that disease incidence among affected cultivars was 13–52% and up to 100% in seriously infected fields. The sugarcane cultivar ‘Yuetang86-368’ was highly susceptible. SCWL is a destructive disease on sugarcane that requires preventive quarantine management measures to prevent its further spread.