AgTech
A scourge of citrus plants is so-called Citrus greening disease, also known as HLB – huanglongbing, which turns fruit sour and kills trees within a few years. It was detected in Florida in 2005, and has turned up in California and Texas, the second- and third-largest citrus producers in the United States. The Asian citrus psyllid, Diaphorina citri, is the vector of citrus huanglongbing disease. These 4-mm-long psyllids, which are related to aphids and whiteflies, transmit the bacterium Candidatus Liberibacter between trees when they feed on new leaves. Several months to 2 years later, as the bacterium damages the vascular system, leaves start to turn yellow (Science, 28 April 2006, p. 523). Trees are infectious even before they show symptoms, which makes it hard to halt the disease’s spread. Only intensive surveillance, relentless removal of sick trees, and aggressive insecticide spraying has been shown to be effective; there is no current cure. The catastrophic effect of insecticide spraying on the bee population, already under colossal environmental pressure, has severely complicated the mitigation effort.
Treatment of citrus greening disease (HLB – Huanglongbin) using the anti-microbial properties of silver nanoparticles.
John recently developed a new Q-switched Nd:YAG laser operating at 1064 and 532 nm as part of a new process for generation of large volumes of high-concentration metal nanoparticles with tight, almost monochromatic size distributions. The high zeta potentials of the silver nanoparticles (AgNPs) formed by this chemical-free technique ensure that they remain in suspension for very long periods of time and can be transported in bulk. These particles are under investigation for their remarkable anti-corrosion properties, their equally remarkable effects on ballistic, hydrodynamic and aerodynamic boundary-layer dynamics, and in medical and life-sciences applications. As part of this project John also developed a multi-chamber flow-cell system for in-line continuous production of nanoparticles that does not use a solid ablation target (which would otherwise need to be periodically replenished) and allows the particle sizes to be tuned and monitored on the fly to optimize for the end-use application.
The anti-bacterial properties of AgNPs are well known, and their incorporation in various materials, surfaces, devices etc. to suppress infection are being studied widely, for example embedded in trach tubes to prevent ventilator-associated pneumonia. Acting on an idea that the bacterium in the psyllid might be killed by AgNPs at a minimum inhibitory concentration (MIC) that might not be toxic to the psyllid itself or eukaryote cells of higher-level organisms, a study of the effect of AgNPs at three concentrations and two mean size distributions on scions infected with HLB was initiated in 2013, with controls comparing the AgNPs to water, DMSO and a more traditional antibiotic (ampicillin).
HLB-infected lemon scions were soaked in solutions of the anti-microbials under test and then grafted on to healthy citrus rootstocks. Samples for DNA analysis were taken some months after inoculation. The results from a preliminary range of nanoparticle sizes and concentrations were very encouraging:
- The Scion Survival Rate % (defined as the number of scions that survived out of the total number grafted – an indicator of phytotoxicity) was 100% in 5 out of the 6 test samples, and superior to the water, DMSO and Ampicillin controls.
- The Scion Growth Rate % (defined as the number of scions that produced new leaves or flushes relative to the total number of grafted scions) showed a distinct AgNP size relationship, with the largest growth rate being somewhat worse than the ampicillin control but comparable to the water and DMSO controls.
- The Scion Infection Rate % (defined as the number of infected scions with Ct value* lower than 36.0 relative to the total number of grafted scions) also showed a clear AgNP size-dependence, with the best-performing size and concentration having 0% infection rate, equal to the ampicillin control.
- The Las transmission rate % [Candidatus Liberibacter asiaticus (Las)] (defined as the number of infected rootstocks with Ct value* lower than 36.0 relative to the total number of inoculated rootstocks) showed a clear AgNP size dependence with all sizes and concentrations being superior to the water and DMSO controls but inferior to the ampicillin control.
- The trend seems to favor AgNP size distributions that are likely to be biocompatible in humans (excreted through the renal system), although it may be possible to introduce the AgNPs into the psyllid in a manner that does not cause the AgNPs to accumulate in the fruit.
- One AgNP size and concentration showed equivalent Ct to ampicillin.*
- Rootstock Ct was generally inferior to ampicillin, but one AgNP size and concentration came close to meeting the 36.0 threshold level.*
The results of this early, preliminary screen with nominal AgNP size and concentrations are quite encouraging. It seems clear that an AgNP concentration and size distribution that simultaneously kills the HLB bacterium but allows insects to survive might well exist. This obviously would have profound implications for the treatment of infected citrus and the well-being of the bee population.
*[Scion Ct and Rootstock Ct are quantitative measures of the presence of HLB observed in the scion and rootstock of the plants. Plants are considered to be infected with HLB in either of these two regions of the plant when real-time qPCR assay has a threshold cycle Ct value less than 36.0. For reference ampicillin shows a Ct of 40.0]