Molecular techniques based on PCR have made it possible to study the diversity of microorganisms in natural environments without culturing (51
). These techniques are valuable for increasing our understanding of microbial communities despite some demonstrated amplification biases due to primer selection, the number of amplification cycles, and template concentration (37
). A diverse assemblage of microorganisms was observed in citrus leaf midribs from HLB disease-positive citrus groves with both PhyloChip analysis and 16S rRNA gene clone library sequencing. The fact that so many more orders of bacteria were detected by PhyloChip analysis (47 orders in 15 phyla) than by cloning and sequencing (20 orders in 8 phyla) indicates that PhyloChip analysis is more comprehensive for identification of microorganisms in environmental samples than 16S rRNA gene clone library sequencing. This is consistent with previous reports that compared clone library data with PhyloChip community analysis data for environmental samples (8
). The PhyloChip array used in this study contains 8,741 taxa representing all 121 demarcated bacterial and archaeal orders (7
). The size of the clone library might also contribute to the difference in the data. It has been suggested that 40,000 sequencing reactions are required to document 50% of the richness of certain environmental samples, which is laborious, costly, and time-consuming (8
). Typical 16S rRNA gene clone libraries include fewer than 1,000 sequences (16
). The numbers of clones obtained for our asymptomatic and symptomatic samples are 957 and 1,105, respectively. The different primers selected for PCR amplification for PhyloChip analysis (primers 27f and 1492r) and construction of a clone library (primers 799f and 1492r) might have contributed to the difference even though both sets of primers include universal primers for bacteria (10
). In addition, it is also possible that the PhyloChip array approach causes nonspecific hybridization, leading to false positives, although this is most likely to hamper discrimination of taxa at the genus or family level (8
). While this may inflate the number of species-level taxa detected per family, it does not affect either the phylum-level richness of the community or the change in relative abundance of “Ca.
Liberibacter asiaticus,” which are the two main points that we meant to address with PhyloChip analysis.
Our study indicated that “Ca
. Liberibacter sp.” is the dominant bacterium that is always detected in citrus showing HLB disease symptoms. 16S rRNA gene cloning and sequencing showed that “Ca.
Liberibacter asiaticus” was the only common bacterium found in all 12 symptomatic trees in two citrus groves. The PhyloChip study indicated that nine taxa were significantly different, and all of them were more abundant in symptomatic plants than in asymptomatic plants. However, “Ca.
Liberibacter asiaticus” was the dominant organism in the symptomatic leaves but not in the asymptomatic leaves, and the observation that “Ca.
Liberibacter asiaticus” was the dominant organism in the symptomatic leaves supports the association between HLB disease and “Ca.
Liberibacter asiaticus” in Florida (5
). By using PhyloChip analysis the otu_7603 taxon, representing “Ca.
Liberibacter asiaticus,” was detected at a very low level in asymptomatic plants, but it was over 200 times more abundant in symptomatic plants. Other than “Ca.
Liberibacter asiaticus,” the taxa which were more abundant in symptomatic plants than in asymptomatic plants included representatives of the taxa Phyllobacter
sp. strains 6904 and 7359, Sphingobacterium
, and Syntrophobacter
(Fig. ), and these bacteria have not been reported to cause plant diseases so far. Their roles in HLB disease symptom development remain to be investigated.
The abundance of some of the bacteria that were detected was greater for asymptomatic samples with HLB disease than for symptomatic samples. For example, incertae sedis 5, Oxalobacteraceae
, and Saprospiraceae
, some of which have biocontrol and plant growth-promoting potential (3
), were found only in asymptomatic samples based on cloning. It is not known whether these bacteria play significant roles in suppressing HLB disease symptoms. The lack of symptoms might in some cases be due to the low titer of “Ca.
Liberibacter asiaticus” in the phloem, considering that previous results indicated that a minimal “Ca.
Liberibacter asiaticus” population is required for symptom development (N. Wang, unpublished data). Interestingly, clone library analysis and “Ca.
Liberibacter asiaticus”-specific PCR suggest that there might be a few escape trees (asymptomatic trees with heavy loads of the putative pathogen “Ca.
Liberibacter asiaticus”). Both methods found “Ca.
Liberibacter asiaticus” in asymptomatic tree G1A4, while clone library analysis also indicated the presence of “Ca.
Liberibacter asiaticus” at high titers in asymptomatic trees G1A1 and G2A5 and PCR showed that “Ca.
Liberibacter asiaticus” was present in asymptomatic trees G1A4 and G2A3. Whether such trees can survive with large populations of “Ca.
Liberibacter asiaticus” without showing any disease symptoms and whether the endophytic microbial community plays a role in symptom suppression remain to be determined.
Citrus leaves can support a diversity of microbes either epiphytically or endophytically. PhyloChip analysis revealed the presence of 47 orders of bacteria in 15 phyla, while 20 orders in 8 phyla were observed with the cloning and sequencing method for the citrus leaf midribs. Actinobacteria
, and Firmicutes
have previously been reported to be associated with plant leaves (23
). The majority of these bacteria are insect transmitted or endosymbionts of insects. “Ca.
Liberibacter asiaticus” has been shown to be psyllid transmitted. Most of the clones in the 16S rRNA gene library were closely related to bacteria reported to be endosymbionts of various insects (17
). Lacava et al. (31
) have reported similarity between the endophytes of host plants and bacteria inhabiting the head region of the glassy-winged sharpshooter, Homalodisca vitripennis
, an important vector of various strains of X. fastidiosa
. Our study also indicates that there may be multipartite interactions between the host plant, the insect vector, and the associated microbial diversity. However, some bacteria, such as Chlamydiae
, AD3, Bacteroidetes
, and mgA-2, have never been reported to be associated with plant leaves (3
). This indicates that our understanding of the extent of microbial diversity associated with plant leaves is still incomplete. It is not surprising that the bacterial population associated with citrus midribs seems to be quite different from and more diverse than the citrus phyllosphere population (52
). The majority of the bacteria in our study are likely endophytes since surface sterilization was used. Surface sterilization has been shown to eliminate most, but not all, microbes on the leaf surface (10
). The microbiome associated with citrus leaves from HLB pathogen-infected groves in Florida is very different from that of X. fastidiosa
-infected citrus groves in Brazil (2
). Curtobacterium flaccumfaciens
, Enterobacter cloacae
sp., and Pantoea agglomerans
were reported for X. fastidiosa
-infected citrus branches in Brazil, while they were not found in our study (2
). This might have been due to differences in the environmental conditions in the two geographic locations where the plants were grown (e.g., geographic areas and weather conditions), dominant pathogens associated with the plants, or the tissues sampled (leaf midrib or branch).
This study included an extensive molecular analysis of the bacteria in citrus leaf midribs from HLB pathogen-positive citrus groves. We demonstrated that both symptomatic and asymptomatic leaves contain a diverse assemblage of bacteria. Some bacteria other than “Ca. Liberibacter” have been identified from citrus with HLB-disease. “Ca. Liberibacter asiaticus” is the dominant organism in the symptomatic leaves compared to the asymptomatic leaves, implicating this organism as the causal agent of HLB disease.