**1. Introduction**

Citrus, including oranges, tangerines, grapefruits, pomelos, lemons, limes, etc., is a perennial grafted crop of the most planted and produced fruits in the world (https://www. statista.com, accessed on 5 January 2023). Moreover, citrus Huanglongbing (HLB) is one of the most crucial and devastating diseases in the citrus industry worldwide. It was included as one of the top ten crop diseases by the Ministry of Agriculture and Rural Affairs of China in 2020. HLB causes the yellowing of new shoots, the mottling of fully mature old leaves, or a zinc-deficiency-like symptom in mature fresh leaves; however, these symptoms occur with no programmed cell death (PCD), such as necrosis. Among these, the mottling leaf is the most typical symptom to identify HLB. Compared to normal healthy fruits, the most reliable diagnostic symptoms of affected fruits are being improperly colored (greening or

**Citation:** Zhang, J.; Liu, Y.; Gao, J.; Yuan, C.; Zhan, X.; Cui, X.; Zheng, Z.; Deng, X.; Xu, M. Current Epidemic Situation and Control Status of Citrus Huanglongbing in Guangdong China: The Space–Time Pattern Analysis of Specific Orchards. *Life* **2023**, *13*, 749. https://doi.org/ 10.3390/life13030749

Academic Editors: Hakim Manghwar and Wajid Zaman

Received: 2 February 2023 Revised: 4 March 2023 Accepted: 5 March 2023 Published: 10 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

'red nose fruit'), being lopsided with a curved columella, and developing mostly aborted seeds. These phenomena of the various symptoms mentioned are particularly prevalent in Guangdong, as HLB ravages the citrus industry there [1–3].

Huanglongbing is caused by phloem-limited bacteria, specifically, the '*Candidatus* Liberibacter spp.' of α-proteobacterium [4]. Three distinct Liberibacter species are related to HLB. The HLB caused by '*Candidatus* Liberibacter asiaticus' (CLas) have a wider geographical spread, are more severe, present in lower elevations, and possess higher temperature tolerance [5]. Further, it is vectored by Asia citrus psyllid (*Diaphorina citri* (Kuwayama)) [6] in a persistent and propagative manner. The HLB caused by '*Ca*. L. africanus' (CLaf) and '*Ca*. L. americanus' (Clam) are more restricted, less severe, and more temperature-sensitive [7]. CLas is the only species detected from HLB-affected citrus in China [8]. After infection, CLas quickly colonizes the root system before canopy symptoms develop. The upward movement of CLas from roots to canopy is linked to seasonal flushes and the CLas population [9]. Globally, HLB has been distributed in 64 countries within Asia, Africa, North America, South America, and Oceania (data accessed on 20 December 2021 from Center for Agriculture and Bioscience International (CABI)). The pathogen, CLas, is widely distributed in Asia and North America, and partially distributed in Africa [10–12]. Though CLam is only partially detected in Brazil [13], there has been a shift in the prevalence of CLam to CLas, which has been recently observed [7].

Citrus cultivation was highly developed early last century in the Pearl River Delta and the Hanjiang Delta in the Guangdong province of China [14]. As determined from the tonnage data of citrus production, the citrus industry peaked in 1933; however, a significant drop occurred in 1934 in several areas, including the Chaoshan area in Hanjiang Delta. According to Lin Kong-Hsiang's publications [1], the HLB epidemic in the Chaoshan area could be traced back to as early as the 1870s, despite its, then, slight prevalence status. Whereas the first report of this disease was most likely recorded by Reinking [15] after a disease survey along the Pearl River Delta and West River, it became a serious disease from the 1930s onwards in Guangdong, with the citrus production cycle becoming shorter as a result [1,14–19]. The report and incidence of HLB in the Fujian and Guangxi provinces was a little bit later than those found in Guangdong. Nonetheless, the HLB in these three provinces has been considered the most widely distributed and the most serious since the mid-20th century. From the late 1970s until the 1980s, HLB became a serious issue in Sichuan, Jiangxi, Yunnan, Hainan, and Taiwan. It then spread into Hunan, Guizhou, and Zhejiang [19] after 1980. Until now, 11 of the 19 citrus cultivation provinces and regions in China, accounting for more than 80% of the total citrus cultivation area, had been damaged by HLB [19].

In recent years, the damage caused by HLB in the Guangdong Province has been increasing; further, it is most likely that the shortened citrus production cycle from planting to replant in the whole orchard is a reflection of this fact. The citrus producers summarized a famous 'ten-year cycle', which means that the trees in the orchards would be replaced every 10 years. This would be performed because most trees were affected and, thus, were producing limited valuable fruits. Take the famous Yangcun citrus farm in Huizhou, which is the largest (2000 hm2) citrus farm in Asia and was established in 1951–1953, as an example. The farm experienced two large-scale disease tree eradications from 1979 to 1982 and during 1996–1999. The production of the fresh fruits reached its peak in 1977 and 1991, but dropped significantly in 1982 and 2000 [17]. Since the ravages of HLB from the 1970s, the citrus production in this area cannot avoid the 'ten-year cycle'. After that, the most famous local varieties, e.g., the *Citrus reticulata* 'Shatang' tangerine in Sihui, the *C. sinensis* Osbeck cv. 'Hongjiang' orange in Zhanjiang, and the *C. reticulata* Blanco 'Tankan' in Puning, were also almost destroyed at the beginning of this century. A recent survey in 2016–2017 concluded that citrus groves in all 17 cities and 65 counties in the Guangdong Province were affected by HLB, and around 59,700 hectares of citrus were affected, accounting for about 25% of the total citrus planting area [20]. Currently, HLB still seriously restricts the development of the citrus industry in Guangdong. However, with more attention being

paid recently via government policy toward the comprehensive prevention and control of HLB, the resumption of citrus production has been promoted. More growers prefer large-scale groves, where standardized management with higher technical specifications is adopted.

Recorded systematic investigation on the occurrence of HLB in the field were mainly concentrated around the 1950s and 1980s [17]. Most of those studies focused on the annual increase and removal of diseased trees in orchards. A few of these investigations analyzed the correlation between geographical environment conditions and the prevalence of HLB. Quite a few studies have analyzed the relationships between HLB prevalence results, and cultivation and management measures. With the development of molecular biology technology, after the pathogen of HLB was identified as a type of bacteria [4], the research hotspots have shifted from disease epidemic to the molecular interaction among the pathogen–host–vector. Although HLB has been ravaging the citrus industry of Guangdong Province continuously, no studies have systematically investigated the annual prevalence of HLB within specific orchards in the last 30 years.

In recent decades, mathematical models play an important role in understanding the epidemiology of HLB [21–24]. However, most of the dynamic behaviors of these models are studied by only using computer simulations or are only understood by professional persons. In this study, space–time dynamic (year-by-year) point pattern measures were applied to highlight the HLB progression over time in the groves of Guangdong. All the screened orchards were larger than 5 hectares and with different management levels. A 'two-to- three year' survey was conducted for the appearance and degree of HLB symptoms on each tree within these groves for the appearance and degree of HLB symptoms. In this study, the aims are to provide guidance for the early warning of HLB, to enhance the prevention awareness of growers, and to provide suggestions on the specific effective measures for the prevention and control of HLB in areas where HLB is severely endemic.

#### **2. Materials and Methods**

### *2.1. Orchard Information and Survey Methods*

To reduce the impact of spatial heterogeneity, the research area is located in South China, specifically in the province of Guangdong. Six orchards in Zhaoqing City, Huizhou City, Guangzhou City, or Meizhou City were selected as the survey sites. Only one Liberibacter species (CLas) was detected in these areas. The tree density of the orchards was similar in all orchards, except for orchard 1, wherein the cultivar within it was usually planted at a high density. The information on the selected orchards is shown in Table 1. The A1 region of orchard 4 was under semi-natural conditions in a screen house. Orchard 5 and orchard 6 contain both sloping plots and non-sloping plots. In the conventional management orchards, the diseased trees were not timely monitored or rouged, and attention was not paid to killing the psyllid. Comprehensive control is taken to mean integrated management concepts, which combine cultural, chemical, and biological control measures that are conducted between 9 and 12 times in a timely manner with pesticide application.

Investigations were carried out via visual inspection in Autumn and Winter from 2019 to 2021, when the diseased citrus varieties showed their apparent symptoms (Figure A1). During each survey, 12 experienced Citrus Huanglongbing Research Laboratory members participated, with 2 persons in a group. Each investigator collected at least 20 leaf samples which were visually inspected as HLB–affected. Further, another 20 samples evaluated as healthy were collected each day. DNA was extracted from the randomly collected samples and amplified by RT-PCR for CLas detection [25]. If the PCR results of 95% of the collected samples were confirmed to be consistent with the visual inspection, the data from the survey were used for further analysis. Otherwise, the surveyed plot would be assigned to another pair of investigators. In addition, the CLas-positive samples were assigned for genetic diversity analysis by conventional PCR and RT-PCR based on the phage types (three type-specific prophage loci), a miniature inverted-repeat transposable element (MITE) region (CLIBASIA\_05620 ~ CLIBASIA\_05625), and their short tandem

repeat genes (CLIBASIA\_03080 and CLIBASIA\_01215) [26]. After collecting the band information of conventional PCR or Ct values of RT-PCR according to Zheng et al. [26], data were further used for diversity analysis of the CLas strains. Cluster analysis of the CLas populations in the six orchards was completed by Popgene v. 1.32 (https://www. ualberta.ca/~fyeh/popgene.html, accessed on 3 March 2023) based on Nei's (1972) genetic distance. The cluster map was generated in MEGA v. 11.0 [27].


**Table 1.** Information of the surveyed citrus groves.

The scales (0, 1, 2, and 3) of the diseased trees were recorded based on the severity of the disease, wherein scale 0 indicates non-HLB-affected trees and scale 1 indicates that 1/3 of the canopy is affected by HLB, etc. The 'two-step-path' app (Shenzhen 2bulu Information Technology Co., Ltd., Shenzhen, China) was applied to record the path/track of the survey, to map the diseased trees in space and to record the time of the surveys. The data from the 'two-step-path' app were viewed and exported by a LocaSpaceViewer4 PC (Zhongke Tuxin Technology Co., Ltd., Suzhou, China). These procedures resulted in the development of space–time point pattern survey maps of the different scaled symptomatic citrus. Counting was carried out in each survey for the total number of trees, the number of those which were removed, and the number of infected trees. The incidence of the latter three was calculated by normalizing the counts to the total number of plants in the orchard.
