The ISME Journal (2012) 6, 363–383 & 2012 International Society for Microbial Ecology All rights reserved 1751-7362/12 www.nature.com/ismej ORIGINAL ARTICLE Huanglongbing alters the structure and functional diversity of microbial communities associated with citrus rhizosphere Pankaj Trivedi1, Zhili He2,3, Joy D Van Nostrand2,3, Gene Albrigo4, Jizhong Zhou2,3 and Nian Wang1 1Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL, USA; 2Department of Botany and Microbiology, Institute for Environmental Genomics, University of Oklahoma, Norman, OK, USA; 3Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA and 4Citrus Research and Education Center, Department of Horticultural Science, University of Florida, Lake Alfred, FL, USA The diversity and stability of bacterial communities present in the rhizosphere heavily influence soil and plant quality and ecosystem sustainability. The goal of this study is to understand how ‘Candidatus Liberibacter asiaticus’ (known to cause Huanglongbing, HLB) influences the structure and functional potential of microbial communities associated with the citrus rhizosphere. Clone library sequencing and taxon/group-specific quantitative real-time PCR results showed that ‘Ca. L. asiaticus’ infection restructured the native microbial community associated with citrus rhizosphere. Within the bacterial community, phylum Proteobacteria with various genera typically known as successful rhizosphere colonizers were significantly greater in clone libraries from healthy samples, whereas phylum Acidobacteria, Actinobacteria and Firmicutes, typically more dominant in the bulk soil were higher in ‘Ca. L. asiaticus’-infected samples. A comprehensive functional microarray GeoChip 3.0 was used to determine the effects of ‘Ca. L. asiaticus’ infection on the functional diversity of rhizosphere microbial communities. GeoChip analysis showed that HLB disease has significant effects on various functional guilds of bacteria. Many genes involved in key ecological processes such as nitrogen cycling, carbon fixation, phosphorus utilization, metal homeostasis and resistance were significantly greater in healthy than in the ‘Ca. L. asiaticus’-infected citrus rhizosphere. Our results showed that the microbial community of the ‘Ca. L. asiaticus’-infected citrus rhizosphere has shifted away from using more easily degraded sources of carbon to the more recalcitrant forms. Overall, our study provides evidence that the change in plant physiology mediated by ‘Ca. L. asiaticus’ infection could elicit shifts in the composition and functional potential of rhizosphere microbial communities. In the long term, these fluctuations might have important implications for the productivity and sustainability of citrus- producing agro-ecosystems. The ISME Journal (2012) 6, 363–383; doi:10.1038/ismej.2011.100; published online 28 July 2011 Subject Category: microbe–microbe and microbe–host interactions Keywords: Candidatus Liberibacter asiaticus; ecosystem functioning; GeoChip 3.0; huanglongbing; microbial diversity; nutrient cycling Introduction (Curl and Truelove, 1986; DeAngelis et al., 2009). The diversity of nutrients and plant secondary Plants are the key primary producers in most metabolites present in the exudates allows enrich- terrestrial ecosystems and generally exploit soils ment of specific taxonomic or functional groups of for resources using complex root systems (Dennis bacteria in the rhizosphere (Bais et al., 2006; et al., 2010). The root exudates allow the main- Haichar et al., 2008; Dennis et al., 2010). Microbial tenance of a dynamic and nutrient-rich niche populations in the rhizosphere are immersed in a around the root–soil interface called the rhizosphere framework of interactions known to affect plant fitness and soil quality (Nannipieri et al., 2003; Correspondence: N Wang, Citrus Research and Education Center, Lugtenberg and Kamilova, 2009). They are involved Department of Microbiology and Cell Science, University of in fundamental activities that ensure the stability Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, and productivity of both agricultural and natural USA. E-mail: [email protected] ecosystems. These microbial populations benefit Received 28 February 2011; revised 8 June 2011; accepted 10 June plant growth by increasing the availability of 2011; published online 28 July 2011 mineral nutrients, production of phytohormones, Role of HLB in microbial communities P Trivedi et al 364 degradation of phytotoxic compounds and suppres- genes across a broad range of microorganisms and sion of pathogens (Kent and Triplett, 2002; Bais functions simultaneously (Zhou, 2003; He et al., et al., 2006; Lugtenberg and Kamilova, 2009). 2007, 2010a). For example, GeoChip 3.0 targets Microbes residing in the rhizosphere also have key several thousand genes from diverse species and roles in ecosystems and influence a large number of groups involved in various functions (He et al., important processes, including nutrient acquisition, 2010a; Zhou et al., 2010). This array allows for a nitrogen cycling, carbon cycling and soil formation detailed analysis of the functional profiles of (Nannipieri et al., 2003; Fitter et al., 2005; Cardon microbial communities and is ideal for understand- and Whitbeck, 2007). These considerations demon- ing how these profile changes respond to environ- strate the importance of studying the structural and mental perturbations (He et al., 2010a, b; Zhou et al., functional properties of rhizosphere microbial com- 2010). munities and the ways in which various factors Huanglongbing (HLB) is the most devastating influence bacterial diversity and microbial activity. disease of citrus in many areas of the world (Asia, Nevertheless, much of the basic information regard- North America, South America and Africa). HLB can ing the community structure of rhizosphere micro- debilitate the productive capacity of citrus trees bial community, their principal functions, their with losses of 30–100% reported (Aubert, 1992). relative stability and the forces that govern their HLB is associated with ‘Candidatus Liberibacter continuity is still lacking. spp.’, which are Gram-negative, phloem-limited Owing to the interest in basic knowledge of plant bacteria (Bove´, 2006). The pathogens are transmitted disease and the economic importance of plant by psyllids Diaphorina citri in Asia, North America disease control, a vast amount of research has been and South America and by Trioza erytreae in Africa dedicated to understanding the basis of the specifi- (Halbert and Manjunath, 2004). HLB pathogens city of plant pathogen interactions, how plants could also be transmitted by HLB-diseased plant defend themselves from infection by pathogens materials used for propagation of nursery plants. and the role of plant beneficial bacteria in the Candidatus Liberibacter asiaticus (Ca. L. asiaticus) control of plant pathogens (Montesinos et al., 2002; was found in Florida in 2005 and spread to all Lugtenberg and Kamilova, 2009). Cheatham et al. citrus-growing counties in the state by 2007. HLB (2009) have stated that the effect of plant disease was also found in Louisiana in June 2008, and in goes beyond yield and pointed towards the scarcity South Carolina and Georgia in 2009. HLB has of reports dealing with the potential of plant not been found in other citrus-growing regions in diseases to affect various ecological processes and the United States, such as Texas, California and services. It has been postulated that disruption of Arizona. However, D. citri Kuwayama, the vector of multitrophic interactions in a stable ecosystem ‘Ca. L. asiaticus’, was reported in Texas in 2001 and under the influence of invading phytopathogens rapidly spread throughout the state by 2006. will cause community reorganization and changes Psyllids were also found in California and Mexico in the local feedback interactions (van der Putten in 2009. Importantly, HLB was confirmed in Mexico et al., 2007). However, there is a paucity of knowl- in December 2009. The infected trees are in the edge on the extent to which such community shifts states of Jalisco and Nayarit, which are B750 miles may occur, on the dynamics of changes and on the away from the Mexican border with California. With putative effects regarding the functioning of eco- the presence of vectors in every citrus-growing state, systems. In addition, establishing linkages between HLB poses immediate threat to the whole US citrus microbial diversity and ecosystem functions is even industry (Gottwald, 2010). Our previous studies more challenging (Nannipieri et al., 2003; Fitter have shown that HLB not only affects plant health et al., 2005). but also manifests profound effects on the structure A few studies have examined the effects of plant and composition of the bacterial community asso- pathogens on the structure of plant-associated ciated with citrus (Sagaram et al., 2009; Trivedi bacterial communities (McSpadden Gardener and et al., 2010, 2011). As the diversity and stability of Weller, 2001; Yang et al., 2001; Arau´ jo et al., 2002; the plant-associated bacterial communities heavily Reiter et al., 2002; Trivedi et al., 2010, 2011); influence soil and plant production and ecosystem however, the implication of shifts in diversity on processes (Nannipieri et al., 2003), fluctuations in ecosystem functions are not well understood. The the bacterial diversity could have serious implica-