PCC June July 2021 e

Figure 1: New growth leaves form buffer- (set as mock, left) or SAMP-treated (right) HLB-positive ‘Madam Vinous’ sweet oranges trees.
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extreme temperatures of 60 degrees
C for 20 hours had minimal effect on
MaSAMP, which still retained most
of its bactericidal activity, whereas
Streptomycin lost its antibacterial activity
following the same temperature
incubation. Thus, SAMP is a heat-stable,
plant-derived antimicrobial peptide
that can directly kill Lcr and suppress
CLso in plants.
SAMP suppresses CLas in HLB-positive
citrus trees.
To determine whether MaSAMP can
also suppress CLas in citrus trees,
we used the pneumatic trunk injection
method to deliver the MaSAMP
solution into the HLB-positive trees.
We first tested with eight CLas-positive
Citrus macrophylla with similar
bacterial titer and disease symptoms for
the treatment. At eight weeks following
two doses injected, separated by two
months of MaSAMP injection, the disease
symptoms and the bacterial titer
in all six treated trees were drastically
reduced compared to the mock-treated
(buffer only) plants and one tree with
no CLas detected. We further tested
with HLB-positive ‘Madam Vinous’
sweet oranges and Lisbon Lemon
trees; those appeared to have declining
symptoms and similar CLas titer. After
MaSAMP treatments, the trees had
developed symptomless new flushes,
while mock trees exhibited symptomatic
flushes (Fig. 1). The CLas titer was
reduced in MaSAMP-treated trees,
while it increased in the mock-treated
trees. Taken together, these results
demonstrate across three trials that
SAMP injection can suppress CLas
titer in three different HLB-susceptible
citrus varieties and can cause trees in
declining health to recover.
SAMP treatment safeguards healthy
citrus trees from CLas infection.
Protecting healthy trees from CLas
infection is critical for managing HLB.
The establishment of defense priming
in plants can promote faster and/or
stronger host immune responses upon
pathogen challenges (Brigitte et al.
2017; Zheng Qing and Xinnian 2013).
To determine whether MaSAMP has
priming activity, we applied it by foliar
spray to citrus plants. We found that
MaSAMP applications triggered prolonged
induction of defense response
genes. Thus, SAMP can potentially
“vaccinate” uninfected citrus trees and
induce defense responses to combat
against HLB. To test the protection
ability of SAMP on citrus trees, we
applied the MaSAMP solution or buffer
as mock treatment by foliar spray onto
young, healthy ‘Madam Vinous’ sweet
orange trees. Five days after treatment,
the trees were exposed to ACP carrying
CLas under the “no choice feeding”
condition for 21 days. We treated trees
with MaSAMP solution by foliar spray
every two months subsequently. The
result indicates that MaSAMP-treated
trees have a lower infection rate.
SAMP disrupts the outer membrane
and causes cell lysis of the bacterial cell.
To understand the mechanism of Ma-
SAMP bactericidal activity, morphological
changes of Lcr post-MaSAMP
treatment were observed using transmission
electron microscopy. Application
of 10 μM MaSAMP to Lcr caused
cytosol leakage and the release of small
extracellular vesicles after 30 minutes
of incubation. The Lcr cells were lysed
within two hours of incubation. We
isolated the membrane fraction from
the MaSAMP treated Lcr and detected
the enrichment of MaSAMP in the outer
membrane fraction compared with
the inner membrane fraction. Thus,
MaSAMP likely disrupts mainly the
outer membrane of Lcr and breaks the
bacterial cells, which leads to cell lysis.
SAMP has Low toxicity.
Because SAMP is internalized by citrus,
it is important to test its phytotoxicity.
We injected different concentrations of
MaSAMP solution directly into citrus
leaves and found that MaSAMP has
little phytotoxicity. Furthermore, we
found that MaSAMP can be detected
in fruit tissue of both Australian finger
lime and trifoliate orange by Western
blot analysis and is very sensitive to
human endopeptidase Pepsin, a major
gastric enzyme produced by stomach
chief cells. Thus, MaSAMP in Australian
finger lime has already been
consumed by humans for hundreds of
years and can be easily digested (Figure
2, see page 13). These results suggest a
low possibility of toxicity of SAMP on
citrus and humans, although additional
safety assessment tests are necessary
for regulatory approval.
12 Progressive Crop Consultant July / August 2021