1. Southwest University/Citrus Research Institute of Chinese Academy of Agricultural Sciences, Chongqing 400712, China 2. National Agricultural Technology Extension and Service Center, Beijing 100125, China 3. Chengdu Plant Quarantine Station, Chengdu 610000, China 4. Key Laboratory of Modern Precision Agriculture System Integration Research, China Agricultural University, Beijing 100083, China
Huanglongbing (HLB, citrus greening) is one of the most serious quarantine diseases of citrus worldwide. To monitor in real-time, recognize diseased trees, and efficiently prevent and control HLB disease in citrus, it is necessary to develop a rapid diagnostic method to detect HLB infected plants without symptoms. This study used Newhall navel orange plants as the research subject, and collected normal color leaf samples and chlorotic leaf samples from a healthy orchard and an HLB-infected orchard, respectively. First, hyperspectral data of the upper and lower leaf surfaces were obtained, and then the polymerase chain reaction (PCR) was used to detect the HLB bacterium in each leaf. The PCR test results showed that all samples from the healthy orchard were negative, and a portion of the samples from the infected orchard were positive. According to these results, the leaf samples from the orchards were divided into disease-free leaves and HLB-positive leaves, and the least squares support vector machine recognition model was established based on the leaf hyperspectral reflectance. The effect on the model of the spectra obtained from the upper and lower leaf surfaces was investigated and different pretreatment methods were compared and analyzed. It was observed that the HLB recognition rate values of the calibration and validation sets based on upper leaf surface spectra under 9-point smoothing pretreatment were 100% and 92.5%, respectively. The recognition rate values based on lower leaf surface spectra under the second-order derivative pretreatment were also 100% and 92.5%, respectively. Both upper and lower leaf surface spectra were available for recognition of HLB-infected leaves, and the HLB PCR-positive leaves could be distinguished from the healthy by the hyperspectral modeling analysis. The results of this study show that early and nondestructive detection of HLB-infected leaves without symptoms is possible, which provides a basis for the hyperspectral diagnosis of citrus with HLB.
1st, pre-denaturation 94°C 5 min 2nd, denaturation 94°C 30 s 3rd, primer annealing 60°C 30 s 4th, extension 72°C 45 s 35 cycles 5th, extension 72°C 10 min
Tab.1
Fig.2
Group
HLB-free orchard
HLB-infected orchard
Calibration group
Validation group
Total
Calibration group
Validation group
Total
Normal color leaves
15
5
20
15
5
20
Chlorotic leaves
55
15
70
55
15
70
Total
70
20
90
70
20
90
Tab.2
Fig.3
Fig.4
Fig.5
Fig.6
Fig.7
Leaf surface
Preprocessing methods
g
s2
Calibration data set
Validation data set
Number of samples
Recognition number
Recognition rate (%)
Number of samples
Recognition number
Recognition rate (%)
Upper
Original spectra
658.28
463.28
140
140
100.0
40
36
90.0
9-point smoothing
599.70
249.04
140
140
100.0
40
37
92.5
D1
0.30
325.40
140
138
98.5
40
34
85.0
D2
1.90
160.30
140
140
100.0
40
36
90.0
MSC
0.76
451.43
140
138
98.5
40
35
87.5
SNV
0.75
511.31
140
138
98.5
40
33
82.5
Lower
Original spectra
0.20
225.05
140
137
98.0
40
36
90.0
9-point smoothing
0.17
157.95
140
137
98.0
40
37
92.5
D1
0.51
126.82
140
140
100.0
40
34
85.0
D2
73.14
107.28
140
140
100.0
40
37
92.5
MSC
35.78
845.29
140
140
100.0
40
36
90.0
SNV
28.94
421.64
140
140
100.0
40
36
90.0
Tab.3
Leaf surface
Pre-processing
Data set
Actual classification
Model classification
Recognition rate/%
Total recognition rate
HLB-negative
HLB-positive
Upper
9-point smoothing
Calibration
HLB-negative
70
0
100
100
HLB-positive
0
70
100
Validation
HLB-negative
20
0
100
93
HLB-positive
3
17
85
Lower
D2
Calibration
HLB-negative
70
0
100
100
HLB-positive
0
70
100
Validation
HLB-negative
20
0
100
93
HLB-positive
3
17
85
Tab.4
Type of leaf
Upper surface spectra+ 9-point smoothing
Lower surface spectra+ SNV
Actual
Model
Actual
Model
Actual
Model
Actual
Model
Chlorotic
15
15
15
13
15
15
15
13
Normal color
5
5
5
4
5
5
5
4
Tab.5
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