Introduction

Sugarcane (Saccharum sp. hybrid) is one of the major tropical C4 plant crops that is cultivated in the tropical and subtropical regions globally and contributes to 75 % of the world’s sugar (Singh et al. 2011). Guangxi is a key player among the sugarcane and sugar-producing provinces of China (Li 2003, 2005). Pokkah boeng disease of sugarcane (a Javanese term), which is caused by Fusarium, was first recorded in Java by Walker and Went in 1886 (Siddique 2007). It was also found that the disease is one of the major constraints in sugarcane production (Singh et al. 2006; Vishwakarma et al. 2013), and is becoming predominant among the foliar diseases in China during the recent years (Wang et al. 2016). This differs from one region to another, and the occurrence of the disease has been recorded in almost all the cane growing countries in the world (Martin et al. 1989; McFarlane and Rutherford 2005; Siddique 2007; Khani et al. 2013; Lin et al. 2014).

Three to seven months old sugarcane is more susceptible to infection than the plants in later stages of growth. As a consequence of infection, the leaves become crumpled, twisted, and shortened accompanied by the malformation or distortion and causes direct losses in yield and juice quality (Whittle and Irawan 2000; Govender et al. 2010). Pokkah boeng disease of sugarcane is an air-borne disease, and the disease response shifts from resistant to susceptible has repeatedly been observed (Lyrene et al. 1977; Chen et al. 2011). It was recorded in India in many varieties: Co419, Co7219, Co7527, CoC 671, Co7527, Co86032, Co8014 (Patil 1995; Vishwakarma et al. 2013) and in China: GT03-3089, GT06-400, GT06-1023. Hence, to explore the establishment of field grading resistance standard for sugarcane pokkah boeng, its occurrence on 11 new sugarcane varieties and the dominant cultivar ROC22 in Guangxi of China were investigated from Nanning, Liuzhou, Hechi, Baise, Beihai, and Chongzuo areas in 2015 (Wang et al. 2016). However, till date, limited information concerning the resistance of sugarcane germplasm resources and the pathogen variability related to host reactions is available in subtropical China.

Therefore, the present investigation was undertaken with an aim to assess the inoculation method for the resistance of pokkah boeng disease for inhibition of Fusarium in sugarcane under greenhouse and field conditions. The new grading standard can be used for rapid and accurate evaluation of the resistance and the regional differences of pokkah boeng disease on sugarcane varieties.

Materials and Methods

Genotype Panels

Three sugarcane clones provided by Guangxi University, i.e. GZ1, GZ2, and GZ3, and a dominant commercial variety ROC22 in China were used for evaluating the artificial inoculation method, and a total of 88 sugarcane genotypes including commercial hybrids, interspecific hybrids, and some clones of basic species of Saccharum (Table 1) provided by GXAAS were assessed for the resistance of pokkah boeng disease.

Table 1 Grading standard of pokkah boeng disease of sugarcane
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Experiment Design

Sugarcane stalks were cut into single buds and planted in the sand-soil of water bucket (35 × 28 × 35 cm) in the greenhouse of GXU. Experiment with the inoculation methods consisted of five treatments (including the controls CK) that were carried out on June 19, 2013. The preliminary resistant assessment of the 88 varieties (clones) was performed in the greenhouse on April 5, 2014, and the further identification of the resistance was conducted in the field on June 8, 2014, in an area specifically known for low pokkah boeng disease incidence in Nanning, Guangxi. All treatments were in a completely randomized blocks design with three replications. Each replication had 20 pots and a total of four buds in each pot. The greenhouse temperature was maintained at 25–35 °C and the humidity at 80–85 %. The chemical properties of the soil were: nitrogen 60.0 mg/kg, available P 62.4 mg/kg, available K 110.5 mg/kg, and organic matter 1.85 %. All materials were adequately planted and covered with plastic films in order to promote the emergence of the buds, and all the agronomical practices were followed except the plant protection measures at the required growth stages.

Fungus Source

Two most virulent and prevalent species associated with pokkah boeng disease of sugarcane, F. verticillioides CNO-1 and F. proliferatum YN-41 in the subtropical China (Lin et al. 2014), maintained and conserved in Guangxi University were used in the present study.

Manual Inoculation

The conidia from the low-temperature maintained culture were used for the inoculation. The budding of conidia was checked before inoculation and multiplied on oatmeal agar (oatmeal 40 g, agar 10 g, water 1 L) for 6–7 days, and conidial suspension (106 conidia mL−1) was prepared in sterile water.

Inoculation Method

Syringe Inoculation

Conidial suspensions of the isolates (CNO-1, 106 conidia mL−1, 100 μL) were microinjected into each sugarcane stalk of four varieties (clones) with a sterile needle during the 4–5 leaf stage. Symptoms were observed on the inoculated stem at 24-h post-inoculation.

Spindle Inoculation

Conidial suspensions of the isolates (CNO-1, 106 conidia mL−1, 100 μL) were dripped into the young spindle, and the symptoms were observed on the inoculated stem in 6–8 days post-inoculation.

Sett Dip Inoculation

Sugarcane stem was immersed in spore suspensions (CNO-1, 106 conidia mL−1, 30 L tank−1) for 12 h and kept under the shade before planting. The disease incidence was investigated after emergence.

Detached Leaf Assay Inoculation

At least five leaves were cut from each variety (clone) with 8–10 cm length by sterile scissors and inoculated in the laboratory with the conidial suspensions of the isolates (CNO-1, 106 conidia mL−1, 100 μL). The length of the lesion was measured at 48-h post-inoculation.

Investigation Method

Bud numbers were counted after emerging and the first occurrence of pokkah boeng disease of sugarcane after a week till the end of the disease infection. The pokkah boeng disease development was monitored by both inoculation methods symptoms (Table 1).

Classification of Resistance Against PBDS

The standard of grade 0–5 was applied to evaluate the resistance of sugarcane varieties against pokkah boeng disease (Wang et al. 2016). Based on the disease severity index (DSI) of pokkah boeng disease of sugarcane, grade 0 highly resistant (HR) was defined as DSI ≤ 1.0; grade 1 resistant (R) as DSI 1.1–5.0; grade 2 moderately resistant (MR) as DSI 5.1–10.0; grade 3 moderately susceptible (MS) as DSI 10.1–15.0; grade 4 susceptible (S) as DSI 15.1–20.0; and grade 5 highly susceptible (HS) as DSI ≥ 20.0 (Wang et al. 2016). DSI was calculated using the following formula:

$${\text{DSI}} = \sum \left( {n_{i} \times v_{i} } \right)/N \times 100\;\% ,$$

where n is the number of sugarcanes with a certain grade, v is the grade, and N is the total number of sugarcanes observed.

Data Analysis

The disease scorings obtained from the different genotypes were statistically analyzed to estimate the resistance as described below.

Results

Effects of Different Inoculation Methods

Through systemic and careful investigation, the symptoms of pokkah boeng disease were recorded after 24-h syringe inoculation (Fig. 1a), the dynamics of the DSI was expressed as a parabolic curve for each variety (clone) and least DSI was recorded in the control treatments. Firstly, the leaf sheaths developed irregular necrotic areas of reddish color, followed by an infection that may form long lesions. Sometimes, the infection in the stem continued upward toward the grow point and affected the stems in severe cases, wherein the entire top of the plant gradually perished. Consequently, the highest DSI value of each community wound appeared after nearly 15 days in four varieties (clones) (Fig. 2a). The ZG1 showed the strongest resistance followed by ROC22 and ZG2, whereas the ZG3 had the worst resistance.

Fig. 1
figure 1

Disease reactions based on the most virulent and prevalent species of pokkah boeng disease infected with 106 conidia of F. verticillioides. a Disease index (DSI) of sugarcane infected with syringe inoculation. b DSI of sugarcane infected with spindle inoculation. c Lesion length of sugarcane with detached leaf assay inoculation. d DSI of sugarcane infected with sett dip inoculation. The test varieties (clones) were ZG1, ZG2, ZG3, and ROC22, respectively

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Fig. 2
figure 2

Symptoms of pokkah boeng disease of sugarcane with syringe inoculation (a), spindle inoculation (b), and detached leaf assay inoculation (c), respectively. The preliminary assessment of the resistance under greenhouse condition

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The spindle inoculation was conducted consecutively (Fig. 1b), the pathogen moves downward into the grow point, and chlorotic or the slightest etiolation symptom on the youngest leaf sheath would be recorded after 6–8 days. Then, the area of lesion and the number of affected leaves gradually increased every day, sometimes narrower or shorter than the normal leaves, or distinct wrinkling and twisting, and the development of the reddish areas into lens-shaped holes, or may form ladder-like lesions after 15 days. The highest DSI value of each community wound appears nearly at 20 days by this inoculation (Fig. 2b). The dynamic changing rule of the DSI and the resistance rank for each variety (clone) were similar to the syringe inoculation, though the DSI was lesser and no dead samples were found.

The lesion length of the leaves inoculated by detached leaf assay inoculation method was recorded after 48 h (Fig. 2c). The results showed that there was no distinct difference either in the diseased time or the severity among the four inoculations at the young plant stage (Fig. 1c).

The samples with obvious symptoms were not found in ZG1, ROC22, and ZG2, while a modest chlorotic or etiolation symptom at the youngest leaf sheath was observed in ZG3 with sett dip inoculation (Fig. 1d).

The results of the initial evaluation of the resistance under greenhouse conditions with spindle inoculation indicated that the proportion of different patterns of resistance and susceptibility were 34, 38.6 % (susceptible CNO-1 and susceptible YN-41); 32, 36.4 % (susceptible CNO-1 and resistant YN-41); 14, 15.9 % (susceptible YN-41 and resistant CNO-1); and 8, 9.1 % (resistant CNO-1 and resistant YN-41), respectively. Moreover, the results showed that: (1) CNO-1 had higher infection than YN-41 by this inoculation with a success of up to 89.8 %; (2) There were 80 varieties (clones) susceptible to these two pathogens, represented as 90.9 % of all, and CP84-1198, GT94-40, GT05-3846, ROC1, ROC27, YC58-14, YC64-173, YT94-128 belonged to the high-resistant varieties (Table 2).

Table 2 Patterns of resistance and susceptibility of genotypes to CNO-1 and YN-41 under indoor conditions
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Screening of Germplasm Varieties (Clones) for Pokkah Boeng Disease Under Field Conditions

Detectable differences were noticed between indoor and field disease reactions of various germplasm clones for pokkah boeng disease. Under greenhouse condition, eleven varieties viz., F134, F160, GT11, GT37, GT92-66, H38-2915, HOCP00-950, NCO310, PQJ2878, VMC97-41, and YT75-191 were recorded as highly susceptible (HS), susceptible (S), and moderately susceptible (MS) with high DSI. Thirty-five varieties viz., CP01-1372, CP49-50, CP51-21, CP53-17, CP65-357, CP72-2086, CP73-351, CP81-1254, CP84-1198, CP88-1546, CP89-1509, CP89-2143, CZ16, CZ69-58, CZ81-548, GT03-2287, GT05-3846, GT28, GT42, GT94-40, PT43-52, ROC1, ROC27, ROC9, YC58-14, YC62-40, YC64-173, YC65-621, YC90-3, YC96-45, YT83-494, YT89-759, YT94-128, YZ94-375, and ZZ74-141 were recorded highly resistant (HR), resistant (R), and moderately resistant (MR) with light DSI (Table 3). Based on the greenhouse test and field test, GT11, GT37, NCO310, POJ2878, and F134 were identified as the susceptible varieties, whereas CP84-1198, GT05-3846, ROC27, and YT94-128 were the highly resistant varieties (clones).

Table 3 Genotypes behavior to pokkah boeng disease under two different conditions
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Discussion

Artificial Inoculation

The inoculation and disease assessment methods were found similar to those described by Prom et al. (1996). During the inoculation, fungal cultures were prepared with two isolates of Fusarium species, which represent the pathotypes present in China. Subsequently, the sugarcane plants were inoculated by spindle inoculation with approximately 100 µl suspension, 30 days after planting. Disease assessments were conducted after long-term observation under different conditions. This study proved that F. verticillioides conidial suspension at 1 × 106 concentration was the most suitable source since there was a significant increase in the symptoms and DSI of the samples that were similar to the results amplified using fungus-conserved ITS1 and ITS4 primers (Lin et al. 2014).

The pathogen directly infected the tender tissue by syringe inoculation without the natural barrier of leaf sheath and significantly increased the degree of disease, leading to higher incidences (Fig. 1a). Compared with uninoculated control samples, all the inoculated samples had higher DSI and mortality rate, which suggested that there was no resistant variety in the pokkah boeng disease system. Hence, this method can be applied to the further identification of physical expression and genetic analysis with superior stability and repeatability; however, the scale of the sample should be assured.

On the contrary, spindle inoculation is a noninvasive and intuitionistic method with different degree of symptoms. This method may be similar to a natural infection that might be the best choice for identification of the resistance of sugarcane germplasm resources at the young stage (Fig. 1b). Although distinct infection could be recorded by the length of the lesion, it is arduous to extrapolate and predict the underlying mechanism of pokkah boeng disease resistance behavior with detached leaf assay inoculation due to the variable color of lesions and the statistical limitation of small sample numbers (Fig. 1c) in the present study. A previous study demonstrated that the age of the sugarcane plants impacts the sugarcane mosaic virus (Balamuralikrishna et al. 2003) and smut (Olweny et al. 2008) infection. Hence, the assessment of infection for pokkah boeng disease should be carried out in various growth stages to achieve its significance.

When compared to the other methods, suspension immersion exacerbated the injury of the wounded tissue. However, the result indicated that sett dip inoculation was not an ideal treatment for the evaluation of the pokkah boeng disease resistance. This may be explained by the following: (1) the pathogen is air-borne and a vascular parasite and (2) combining with the antagonism between soil microorganism (Aoki et al. 2014), leads to a low incidence of the disease (Fig. 1d).

And the comparison of DSI of tested varieties studied indicated that: (1) CNO-1 had not only higher infection than YN-41, but also higher virulence; (2) the different degree of infection by artificial inoculation showed similar tendency with that of the natural infection. Results of spindle inoculation were very similar to those from syringe inoculation; severe pathogen stress could cause irreversible cellular damages leading to death. Nonetheless, plants are able to withstand periods in a negative status and restart their metabolic functions, and the DSI tends to decrease after reaching maximum under field and greenhouse evaluations; (3) The DSI value of indoor conditions was higher than in the field; and (4) After infecting, both death rate and DSI significantly increased in the susceptible varieties, and the incubated samples had an unreasonable community, all growing worse than the non-incubated plants in similar conditions.

Screening of Resistant Germplasm Resources

For broadening the germplasm base of sugarcane breeding, hundreds of wild germplasm materials including S. spontaneum, S. arundinaceum, and Miscanthus were collected from various locations in China, and more than 300 varieties (clones) were introduced from exotic countries during the past decades. The results of present study revealed that pokkah boeng disease has been largely managed through the deployment of R varieties. When both parents were resistant, a very high proportion of R progenies were obtained, and when one of the parents was S, a moderate number of progenies were R, and when both the parents were S, the proportion of R progenies was less. Thus, for a high level of resistance in the progenies, it is essential to identify and use the R parents.

The selection of suitable parents for the hybridization to produce elite progenies is the key to the success of the sugarcane breeding program (Gazaffi et al. 2010; Hapase 2012). The phylogenetic analysis of POJ canes, CP canes, and Co canes indicated that POJ2878, CP49-50, and Co419 were the widely used parental clones because of their commercial superiority due to high sugar content coupled with high cane yield and superior resistance (Rao 1989; Appunu and Premachandran 2012).

Different conclusions attained in separate studies were due to the distinct results obtained in specific pathogen-host systems. In this study, POJ2878 is one of the original varieties of S. officinarum and a source of high sugar content coupled with high cane yield gene in modern sugarcane varieties. However, pokkah boeng disease with the spread of the commercial variety POJ2878 and its offspring was extensively investigated in sugarcane area worldwide.

The pedigree analysis of the susceptible varieties evolved, viz., varieties F134 (parents Co290 × POJ2878; grandparents Co221 × D74, POJ2364 × EK28), GT11 (parents CP49-50 × Co419; grandparents CP34-120 × Co356, POJ2878 × Co290), GT37 (parents ZZ92-126 × CP72-2086; grandparents ZZ80-101 × ROC1, CP62-374 × CP63-588), NCO310 (parents Co421 × Co312; grandparents POJ2878 × Co285, Co213 × Co244), POJ2878 (parents POJ2364 × EK28; grandparents POJ100 × Katsuya, EK2 × POJ100), revealed that POJ2878 was a susceptible PBDS variety and might result in more PBDS S progenies. The pedigree analysis of the resistant varieties evolved, viz., varieties (clones) CP84-1198 (parents CP70-1133 × CP72-2086; grandparents CP53-63 × ?, CP62-374 × CP63-588), GT05-3846 (parents CP93-1634 × CP90-1424; grandparents are CP canes), ROC27 (parents F176 × CP58-48; grandparents PT58-267 × PT59-2274, CP44-155 × CP50-38), YT94-128 (parents ZZ80-101 × ROC1; grandparents YT54-143 × YC73-226, F146 × CP58-48), revealed that the CP canes exhibited superior resistance to pokkah boeng disease.

Conclusions

The identified varieties (clones) viz., CP84-1198, GT05-3846, ROC27, and YT94-128 found resistance against pokkah boeng disease by spindle inoculation with F. verticillioides can be used for germplasm resource management to broaden the genetic base of the commercial sugarcane, particularly in subtropical China.