(Liberibacter Spp.) Associated with Citrus Greening Disease in Uganda

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Detection and identification of etiological agents (Liberibacter spp.) associated with citrus greening disease in Uganda

A. Kalyebi1, G. Aisu1, I. Ramathani1, J. Ogwang1, N. McOwen2 and P. Russell2

1National Crops Resources Research Institute, P.O Box 7084, Kampala, Uganda 2Agdia Incorporated, 30380 County Road 6, Elkhart, Indiana 46514, USA

Author for correspondence: [email protected]

Abstract

Citrus Huanglongbing (HLB), commonly referred to as citrus greening, is one of the major disease challenges of citrus. In Uganda, there was limited information on Liberibacter spp., the causal organisms of HLB disease, and its psyllid vectors despite the presence of HLB symptoms on citrus. The objective of this study was to identify the Liberibacter spp. and psylla vector species responsible for HLB in Uganda. Other native plant species that could be hosts to pathogens and the psyllid vectors, but are not in the citrus genus, were also investigated. A survey was conducted in 15 citrus growing districts, and symptomatic citrus leaf samples collected, as well as citrus psyllid nymphs and adults for isothermal detection of pathogens in the laboratory. Two types of bacterial pathogens responsible for HLB were detected, namely Candidatus Liberibacter africanus (CLaf), known as the African type; and Candidatus Liberibacter asiaticus (CLas), known as the Asian type. CLaf was found in Mukono and Wakiso districts in Central Uganda and in Mbarara in Western Uganda; while CLas was found in Budaka and Tororo districts in Eastern Uganda. Citrus psyllids, which are the major known disease vectors were present in seven out of the fifteen districts. Psyllid vector identification by morphological means indicated Trioza erytreae, the African psyllid to be the insect vector. Psyllids were common on tangerines (66.7%), Sour Orange (13.3%) and Rough Lemon (13.3%); and least on Washington Navel (6.7%). Three non-citrus plants, Stephania abyssinica (Dill. & A. Rich) walp var. tomentella (Oliv.) Deils (Menispermaceae), Diospyros mespiliformis (Ebenaceae) and Ficus spp (Moraceae) were found to be alternative host plants for the psyllid.

Key words: Candidatus, Diospyros mespiliformis, Psyllids, Trioza erytreae

Introduction 2002). By the 1990s, it was estimated to cover an area of 1,320 hectares, with a Citrus is one of the largest fruit crops total citrus fruit production of about 15,840 grown in Uganda together with mango, metric tonnes. The major citrus growing pineapple, avocado and papaya (UNIDO, districts by then were Kamuli, Soroti and

http://dx.doi.org/10.4314/ujas.v16i1.4 44 A. Kalyebi et al. Lira producing between 2,400 and 3,000 region, mainly in the Teso sub-region tonnes; followed by Mbale, Iganga and (NPA, 2007). A number of varieties of Kumi producing between 1,200 and 1,800 citrus were grown in the country, brought tonnes. from South Africa, Jamaica, Australia, Between 1990 and 2004, citrus Florida and Hawaii in USA, Israel and production began to decline, and based on Spain. The varieties include among others, symptoms, a disease citrus greening was Washington Navel, Hamlin, Valencia, suspected, although not confirmed due to Rough Lemon, Sour Orange, Lime, its resemblance with nutritional disorders, Tangelo, Tangerine and Nova (NaCRRI- and the complexity of conventional NARO, 1988 unpublished report). methods in diagnosing the disease (Dr. J. Citrus Huanglongbing (HLB), Ogwang, NaCRRI, pers. Comm). Due to commonly referred to as citrus greening, suspected citrus greening disease, over the is one of the most serious diseases of citrus years, a lot of citrus plants were removed in the world, responsible for the destruction and some abandoned, while other trees of several citrus industries in Asia and showed signs of recovery from disease Africa (da Graca, 1991). The disease is symptoms. Citrus production in Uganda characterised by yellow shoots, mottling, has since remained relatively low with chlorosis resembling zinc deficiency (Fig. lower than average yields, high production 1), twig dieback and reduced fruit size and and marketing costs and problems quality (Capoor et al., 1974). Fruits do not associated with pests and diseases colour properly (remaining greenish yellow (Sengoba et al., 2002). instead of uniformly yellow), and usually Under the Ministry of Agriculture, have a bitter taste. The disease starts Animal Industry and Fisheries’ small, but with time the number of affected Development Strategy and Investment branches on the orchard tree increases Plan (DSIP), fruit growing, particularly until the whole tree is affected. citrus, is prioritised to improve farmers’ Two species of phloem-limited incomes and nutritional security in the bacteria, Candidatus Liberibacter country (MAAIF, 2010). Survey figures africanus (CLaf) and Candidatus showed that about 13,000 farmers were Liberibacter asiaticus (CLas), are engaged in citrus production in Uganda, considered to be the causal organisms of with a strong concentration in the eastern citrus greening; and more recently, an

Figure 1. Some symptoms of Citrus Huanglongbing (HLB) observed in farmer’s fields in Uganda. (a) zinc-deficiency-like symptoms, (b) severe dieback, both typical of citrus greening disease. and (c) a disease-free citrus plant. Detection and identification of etiological agents associated with citrus greening disease 45 American strain, Candidatus Liberibacter Manjunath, 2004). Psyllids are also known americanus (CLam) was discovered in to inject, during feeding, a salivary toxin Brazil (Texeira et al., 2005). that stops terminal elongation and causes Although grafting has been found to malformation of leaves and shoots play an important role in transmission, (Michaud, 2004). spread of the disease is almost always due Cuscuta (dodder) is also currently to the presence of its vectors, primarily considered a host plant for greening as two species of psyllid insects, the Asian the pathogen can multiply within the plant citrus psyllid, Diaphorina citri successfully and has been used to transmit Kuwayama (Hemiptera: Psyllidae); and CLas and CLaf (Garnier and Bove, 1983; the African citrus psyllid, Trioza erytreae Olfato et al., 1991). More recently, dodder (Del Guercio) (Triozidae) (McClean and has also been used to transmit CLam to Oberholzer, 1965; Martinez and Wallace, Nicotiana tabacum L cv xanthi seedlings 1967). The two psyllids are the only known (Francischini et al., 2007). insect vectors of the etiological agents There is a wide presence of HLB on (bacteria) that cause the greening disease. the African continent (Aubert et al., 1988). In nature, CLaf (African HLB) is The Indian Ocean islands of Reunion and transmitted primarily by infected T. Mauritius harbour both forms of disease erytreae in the fields (McClean and (CLas and CLaf) and both psylla, T. Oberholzer, 1965) and by grafting infected erytreae and D. citri. Ethiopia reportedly material onto non-infected material (Lopes has both T. erytreae and D. citri and the and Frare, 2008). CLaf can also be CLaf HLB organism (Hodkinson, 1980); transmitted transovarially by T. erytreae and more recently, the CLas HLB (van den Berg et al., 1991-1992). The pathogen (Saponari et al., 2010). Within Asian citrus psyllid D. citri is responsible the East African region, HLB has been for transmitting CLas (Asian HLB) reported in Kenya (Bove and Garnier, (Martinez and Wallace, 1967; Capoor, 1984); Tanzania (Swai, 1988), Rwanda 1967) and CLam (American HLB) and Burundi (Aubert et al., 1988), with (Yamamoto et al., 2006), with a further CLaf as causal organism and T. erytreae transmission by grafting and propagation as vector. There is no information to date of infected citrus tissues (Li et al., 2006). on HLB and its vectors in Uganda. Early Experimentally, T. erytreae has been detection of the citrus greening disease found to transmit CLas (Mansonie et al., and its associated bacteria in host plants 1976), while D. citri can also transmit and vector insects is, therefore, essential CLaf (Lallemand et al., 1986). There is not only to disease control and no report to date to indicate whether T. containment, but also in reducing its spread erytreae can transmit CLam ever since it to new areas. was discovered in Brazil. Besides their This study was undertaken to detect role in transmission of HLB, psyllid feeding and establish the etiological agents directly causes damage to the citrus and responsible for HLB disease; and the related ornamental crops as they extract associated vector psylla species in sap from the plant, and produce enormous Uganda. amounts of honey dew (Halbert and 46 A. Kalyebi et al. Materials and methods and adults. Young or new flush of citrus and ornamental nursery plants in the target Survey areas and sampling areas, was inspected for presence of Surveys are highly useful to identify foci yellow-orange eggs, nymphs and emerging of infection (Lohr, 1999). Surveys for or emerged adults. Nymphs were citrus greening and citrus psyllids were collected with thin forceps; while adult conducted in major citrus producing psyllids were collected with an aspirator. districts of Uganda between September Newly emerged adults stay close on leaf and December 2012. The districts were surface. A stem was later tapped three Soroti, Serere, Kamuli, Iganga, Tororo, times with a white sheet held below it (Hall Wakiso, Mukono, Budaka, Bukedea, Arua, and Hentz, 2010) and any adults observed Moyo, Mbale, Kumi, Apac and Mbarara. on sheet were collected. Both nymphs and Citrus fields for sampling were randomly adults on symptomatic or asymptomatic selected within the districts and geo- trees, were collected. About 20 psyllids referenced by Geographical Positioning constituted a sample. System (GPS). Citrus fields included Psyllid nymphs and adults were put in commercial nurseries from both local and vials containing 70% ethanol. Identification improved varieties. Visual assessment of of psyllids species was undertaken using symptom severity was made and leaf adult morphological characters (Martin et samples suspected of citrus greening al., 2012). Specimens were deposited at collected. the NARO Insect museum in Kawanda, According to Li et al. (2009), all Uganda for referencing. Some nymphs on phloem-containing plant tissue can be used leaves were allowed to emerge in netting. to sample for Liberibacter infection. In this Additionally, records of temperature and study, only leaves were used as symptom relative humidity in sampled locations were expression easily becomes evident. taken. Scouting for psyllids continued into Leaves from plants showing greening neighbouring fields (plants) to identify symptoms were collected, in individual potential alternative host plants for psyllids plastic bags, and stored in cool boxes at least twice at each site. (room temperature) from which they were dispatched to Agdia Laboratories, USA. Psyllid extraction Twenty leaves (at the exterior of Plant tissue and psyllid samples were branches) were picked selectively around extracted and tested at Agdia, Inc. the tree canopy to ensure sample Centriguation was done at 6000 rpm for 5 representativeness. The number of trees seconds using a benchtop microcentrifuge sampled per site per district was variable (USA Scientific, catalogue number 2641- and depended on severity of symptom 0016). Leaf samples were prepared by expression (more severe symptoms excising sections of the petiole and mid- indicate highly suspect trees) and vein tissue from each leaf in the sample, frequency of citrus fields (Table 1). using a razor blade. Excised leaf tissue was added to Agdia’s proprietary Collection of citrus Psyllids extraction buffer (AMP1) at a 1:5 weight- A search was conducted for citrus psyllids to-volume ratio in a mesh bag (Agdia using two methods, flush observation and #ACC00930); and macerated using a drill stem tapping, targeting particularly nymphs press. Extracts were transferred into 1.5 Detection and identification of etiological agents associated with citrus greening disease 47 Table 1. AmplifyRP results of leaf and citrus psyllid samples examined from each of the sampled districts surveyed in Uganda

District S/No. Sample Variety Liberibacter Liberibacter Liberibacter type americanustest asiaticustest africanustest

Mukono 1 Leaf Sour Orange -ve -ve -ve Kamuli 2 Leaf - -ve -ve -ve Mbale 3 Leaf Wash. Navel -ve -ve -ve Iganga 4 Leaf Tangelo -ve -ve -ve Bukedea 5 Leaf Hamlin -ve -ve -ve Budaka 6 Leaf Rough Lemon -ve -ve -ve Soroti 7 Leaf Wash. Navel -ve -ve -ve Soroti 8 Leaf Valencia -ve -ve -ve Soroti 9 Leaf - -ve -ve -ve Serere 10 Leaf - -ve -ve -ve Serere 11 Leaf Wash. Navel -ve -ve -ve Serere 12 Leaf Valencia -ve -ve -ve Kumi 13 Leaf Valencia -ve -ve -ve Kumi 14 Leaf - -ve -ve -ve Tororo 15 Leaf Valencia -ve -ve -ve Tororo 16 Leaf Tangelo -ve -ve -ve Kamuli 17 Leaf Valencia -ve -ve -ve Kamuli 18 Leaf Hamlin -ve -ve -ve Wakiso 19 Leaf Rough Lemon -ve -ve -ve Mbarara 20 Leaf Wash. Navel -ve -ve -ve Mbarara 21 Leaf Valencia -ve -ve +ve Mbarara 22 Leaf Tangerine -ve -ve -ve Mbarara 23 Leaf Hamlin -ve -ve -ve Wakiso 24 Psyllid Rough Lemon -ve -ve +ve Budaka 25 Psyllid Rough Lemon -ve +ve -ve Tororo 26 Psyllid Wash. navel -ve +ve -ve Mukono 27 Psyllid Sour orange -ve -ve +ve Arua 28 Psyllid Tangerine -ve -ve Arua 29 Psyllid Tangerine -ve -ve Arua 30 Psyllid Tangerine -ve -ve Arua 31 Psyllid Tangerine -ve -ve Moyo 32 Psyllid Sour orange -ve -ve Apac 33 Psyllid Tangerine -ve -ve Apac 34 Psyllid Tangerine -ve -ve Apac 35 Psyllid Tangerine -ve -ve Apac 36 Insect spp Tangerine -ve -ve Apac 37 Psyllid Tangerine -ve -ve Apac 38 Psyllid Tangerine -ve -ve Mukono 39 Psyllid S. abyssinica -ve -ve Mukono 40 Leaf S. abyssinica -ve -ve

+ve denotes ‘positive’ while –ve denotes ‘negative’ occurrences. 48 A. Kalyebi et al. ml microcentrifuge tubes, vortexed for 30 and tested successively. The first test seconds, and spun briefly using a bench included 23 leaf tissue and 4 psyllid top micro-centrifuge. samples. The ethanol was removed from psyllid The second test included 1 leaf tissue samples by aspirating using a pipette, and and 12 psyllid samples. Based on the air-dried at room temperature for one hour. results of the initial test, each sample was Agdia’s proprietary extraction buffer tested using the CLaf and CLas (AMP1) and 0.2 mm glass beads were AmplifyRP tests, but not CLam. DNA added to each sample and macerated with purified from known CLaf, CLam, or a pellet pestle (Fisher Scientific Co., CLas-infected leaf tissue was mixed with catalogue number 12-141-363). Psyllid a crude extract prepared from healthy samples were homogenised by vortexing lemon leaf tissue to demonstrate crude for 30 seconds and spun briefly using a extract and AMP1 do not inhibit bench top micro-centrifuge. AmplifyRP isothermal reactions.

Pathogen detection Results Leaf and psyllid samples were tested for Candidatus Liberibacter asiaticus (CLas), Liberibacter spp. and distribution Ca. Liberibacter africanus (CLaf), and The leaf and psyllid samples revealed the Ca. Liberibacter americanus (CLam) presence of Candidatus Liberibacter using Agdia’s new Acceler8® AmplifyRP® asiaticus in Eastern (Tororo, Budaka) and isothermal amplification platform (Sutula Candidatus Liberibacter africanus in the et al., 2010; Russell et al., 2011). western (Mbarara) and central regions AmplifyRP utilises recombinase- (Mukono, Wakiso), in Uganda. CLam was polymerase amplification (Piepenburg et not traceable. The two pathogens detected al., 2006) to promote amplification of are known to be responsible for Asian and target DNA sequences that are labeled African citrus greening disease, with markers during the amplification respectively. All leaf samples (except from process, and then detected on lateral flow Mbarara) tested negative for Candidatus strips. AmplifyRP has been compared Liberibacter spp., in spite of showing with qPCR and results show equivalency symptoms of greening in the field (Table in detection (P. Russell, unpublished data). 1). Citrus varieties from which leaf AmplifyRP is sensitive, effective and has samples were collected were Tangerine the specificity of PCR-based testing (45.4%), Valencia (18.2%), Washington methods (Sutula et al., 2010). Extracted Navel (12.1%) and Rough Lemon samples were tested for the presence of (12.1%), Hamlin (9.1%) and Tangelo liberibacter, by adding leaf or psyllid crude (6.1%). Relatively more psyllid samples extract to CLas, CLaf, and CLam (Wakiso, Mukono, Budaka and Tororo) Acceler8 AmplifyRP reactions, and then tested positive for Candidatus tested for liberibacter DNA on lateral flow Liberibacter spp. Psyllid samples from detection strips according to Arua, Moyo and Apac districts tested manufacturer’s specifications. Results negative for Candidatus Liberibacter spp were determined by visually analysing the (Table 1). Adult morphological characters strips. Leaf tissue and psyllid samples examined, especially the pale green were received on two separate occasions appearance upon emergence and the Detection and identification of etiological agents associated with citrus greening disease 49

Figure 2. Some non-citrus host plants of a Psyllid vector of Citrus Huanglongbing (HLB) in Uganda. (a) Stephania abyssinica (Menispermaceae) showing pit-galling, leaf distortion and yellowing symptoms as a result of damage by psyllid nymphs, T. erytreae, (b) symptoms of nymph damage on citrus plant, (c) the Jackalberry tree, Diospyros mespiliformis (Ebenaceae), and (d) the Fig tree, Ficus spp (Moraceae), all show symptoms of psyllid pit-galling. brown/black colour of antennae, feet and (Ebenaceae) and the Fig tree, Ficus spp head indicated that the psyllid species was (Moraceae) (Fig. 2) were found to host the African type Trioza erytreae. psyllids and, hence, identified as alternative In general, although the symptoms of host plants for T. erytreae. Stephania the disease were evident in almost all abyssinica was recovered in Mukono locations surveyed, samples of leaves and district with symptoms of psyllid feeding nymphs collected from all other locations, damage (pit-galling, leaf distortion and except Rubongi, (Tororo), Naboa yellowing of affected foliage) (Fig. 2), with (Budaka), MbaZardi (Mbarara), Kavule nymphal stages breeding on it. The plant (Mukono) and Kawanda (Wakiso), were was recovered in a bush closer to citrus negative for both types of pathogens. fields. Results for this plant were negative Generally, the incidence of citrus for the two species (CLaf and CLas) psyllids was low and psyllids found in 7 tested, despite harbouring both nymphal out of the 15 districts. A total of 66.7% of and adult psyllids. Psyllids were also found psyllids were collected from tangerines, negative for Liberibacter spp. Diospyros 13.3% from both of sour orange and rough mespiliformis with psyllids was observed lemon, and only 6.7 % from Washington in Opuyo, Soroti district; while Ficus spp Navel. Mostly, nymphal stages were was found in Kawanda in Wakiso district. observed and collected. Adults were observed in most cases soon after Discussion emergence, closer to the nymphal case (shell). Citrus psyllids were found only in The present study reveals that not only is Apac, Tororo, Budaka, Arua, Moyo, the African pathogen (CLaf) present, but Mukono and Wakiso districts. that the Asian pathogen (CLas) is present in Uganda (Table 1). The two pathogens Non-citrus psyllid host plants are responsible for African and Asian Three plants, Stephania abyssinica (Dill. forms of greening, respectively. The Asian and A. Rich) walp var. tomentella (Oliv.) pathogen has been known to be Deils (Menispermaceae), the Jackalberry predominantly Asian in distribution and tree, Diospyros mespiliformis historically endemic to Mauritius and the 50 A. Kalyebi et al. Reunion islands within Africa. More that both T. erytreae and CLaf do not recently, CLas has been detected in tolerate hot and dry climates (Bove and Ethiopia in sweet orange (Saponari et al., Cassin, 1968; Green and Catling, 1971); 2010). To our knowledge, this is the first thus the disease was restricted to cool report on the occurrence of HLB elevated areas, while D. citri and CLas pathogens (CLaf and CLas) in Uganda, can resist a wide range of climatic and the second record of CLas and this conditions, including high temperatures form of greening outside Mauritius and the (Bove and Cassin, 1968; Bove et al., Reunion, within Africa. CLaf is 1974). predominant in Africa, and only found in Although greening in Africa and Saudi Arabia and Yemen in Asia. It is not Yemen is known to be transmitted under clear how the pathogen might have moved natural conditions by T. erytreae to Uganda but with the ever increasing (McClean and Oberholzer, 1965; EPPO/ international trade, travel and immigration, CABI, 1996a); and in Asia by Diaphorina the probability of unintentional introduction citri (Capoor et al., 1967; EPPO/CABI, is high. Control of this pathogen and, 1996b), it is possible that either species therefore, HLB disease is usually through could transmit either of the pathogens quarantine to make sure that the bacteria (Manssonie et al., 1976; Lallemand et al., is not introduced or established (da Graca 1986). Further screening and identification and Korsten, 2004). of foci psyllids from a wider area needs Given their nature, the observed to be undertaken to rule out the possible distribution of the two pathogens (CLaf introduction of D. citri. The activity, the and CLas) in Uganda may be influenced population dynamics and population by climatic conditions particularly genetics of both the psyllid and liberibacter temperature. Whereas CLas was species also need to be understood. detected in Eastern Uganda where it is The fact that many leaf samples tested relatively hot, CLaf was found in western negative, in spite of showing symptoms and central Uganda where conditions are of greening, may be due either to lower relatively warm and cool. Average concentrations or uneven distribution of temperatures in Eastern Uganda range pathogens within citrus tissues or hosts from 27 to 35 oC while that of central and (McClean, 1970; Huang, 1979). Typically, western region from 25 to 30 oC, if a plant is infected and displays respectively. These temperature symptoms, then the titer of bacteria is high differences could easily be responsible for enough to be detected by AmplifyRP. distribution of pathogens and disease as Uneven distribution of pathogens previous work has shown CLam, CLaf, sometimes makes detection difficult while and CLas have different temperature in some cases, nutritional deficiencies also tolerances. CLas is capable of thriving at appear to resemble HLB-infection temperatures >35 °C, while CLam could symptoms and various tests need to be not be detected in plants maintained in carried to confirm. temperatures above 32 °C (Hoffman, et. Based on severity of symptoms, al., 2013). CLaf is particularly greening was much more severe in temperature sensitive and thrives only in Eastern (e.g. mean severity 4.6 in Mbale, a narrow range of 22-25 °C. This 3.1 in Serere) than Central and Western corroborates with earlier studies that found (1.6 in Iganga, 2.7 in Mbarara). This Detection and identification of etiological agents associated with citrus greening disease 51 relates well with previous findings that Kasambula and James Nsubuga during CLas is more severe than CLaf although field surveys. Thanks to Deborah Groth- disease severity may be a function of other Helms at Agdia, for the prompt processing factors such as nutritional status of the and testing of samples, and to Olivia soil, age of the trees and whether one Wanyana of Makerere University, strain or a combination of pathogens is Department of Botany, for identifying the present. There was no plant or single host plant samples. location where the two pathogens were found to be co-existent. 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