A grapefruit tree on sour orange rootstock in a residential property in Mission, TX was suspected to have citrus dry root rot disease based on symptoms. The causal organism was isolated from the infected root samples and based on fungal cultural and microscopic morphology and PCR, it was confirmed to be Fusarium solani (Martius) Appel & Wollenweber emend. Snyder & Hansen. A total of 10 healthy sour orange rootstock seedlings were inoculated using conidial suspension of the fungus by the standard root-dip method. After 9 month post inoculation, the inoculated fungus was re-isolated from root and stem sections of these plants. Plants were smaller in size and displayed the classical symptoms of dry rot. The fungal colonies were confirmed to be F. solani based on fungal morphology and PCR. This is the first report of F. solani infecting sour orange rootstock plants in Texas.

RNA sequencing (RNA-seq) methods provide a complete description of RNA transcripts including alternative splicing and small RNA characterization. RNA-seq performed in our laboratory on healthy and Huanglongbing (HLB)-infected young leaves revealed that 4,044 transcripts were up-regulated and 2,562 were down-regulated in the diseased trees. Moreover, a number of genes showed alternative splicing events including exon skipping, intron retention, and 5' and 3' alternative splicing. Furthermore, quantitative PCR (qPCR) performed on 20 randomly chosen genes with high differential expression (10 up and 10 down regulated) showed that all were consistent with RNA-seq data. Additionally, variation in levels of gene expression was observed between young and mature leaves. These early host plant response genes due to HLB-infection might be useful in the development of early HLB-detection methods before manifestation of disease symptoms in the infected plants.

A total of 108 root and corresponding symptomatic leaf samples from four different quadrants were collected from 27 6 year old sweet orange trees in which the presence of Candidatus Liberibacter asiaticus (CLas) was previously confirmed by quantitative polymerase chain reaction (qPCR). There was no significant difference (p>0.05) in the level of infection as determined by threshold cycle (Ct) values between different types of tissue tested. Analysis of variance (ANOVA) showed that there is no significant difference (p>0.05) in test results among different distances from the trunk or quandrants where the root samples were collected. The stumps of the infected trees were covered in a psyllid-proof cage and leaves from the new shoots emerged from these stumps did not show the presence of CLas where as the roots showed the presence of CLas.  Moreover, there was no significant difference between roots from infected trees and roots from infected stumps.  Additionally, there was a significant difference with an average Ct value difference of 2.97 cycles between the DNA samples extracted from roots using two different commercially available kits.

Now that the presence of Huanglongbing (HLB) has been confirmed in California, protecting the state’s citrus industry through early detection of disease is essential in curtailing its spread. Because ‘Candidatus Liberibacter asiaticus’ (Las), the putative causal agent of HLB, accumulates in its vector, the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama, accurate testing of the insect is vital. Due to the fact that insect secondary metabolites interfere with downstream applications (1, 3) there is concern about the number of insects pooled in DNA extractions without compromising Las detection. The current USDA CPHST approved method of testing (2) limits the pooling size to five individual insects per extraction followed by the highly sensitive quantitative polymerase chain reaction (QPCR) detection technique. The QPCR reaction targets a region of the Las 16S ribosomal gene (4) and simultaneously the D. citri specific wingless (WGLS) gene (5) as an internal control for extraction efficiency. In contrast, the CDFA laboratory routinely pools up to 25 individual insects for DNA extraction, followed by the same QPCR detection. If pooling 25 individuals is indeed a safe practice all the labs currently limiting sample pooling to five individuals could save a substantial amount of time and money. We decided to approximate the pooling limit using single insect equivalents of DNA extracted from Las infected ACP from colonies at USDA-ARS in Fort Pierce, Florida pooled with intact ACP from clean UC Riverside, California, quarantine colonies. Depending on results we would follow these experiments with experiments using intact Las positive ACP.