Introduction
Food security and increased global population have driven agricultural activities
to strive for higher productivity. There are however numerous invasive plant pathogens
that cause plant diseases and reduce crop yields, including viruses, fungi, nematodes,
mycoplasma, bacteria and others (Raigond et al., 2022; Chikh-Ali and Karasev, 2023;
Thakur et al., 2023). Approximately, $220 billion is lost to the global economy each
year as a result of plant diseases caused by these pathogens (Lal et al., 2021; Tiwari
et al., 2021; Raigond et al., 2022). Diseases of plants are historically diagnosed
by looking at their symptoms and appearance, often at advanced stages when they are
difficult to manage and treat. Single or mixed infections can occur with pathogens
(Shah et al., 2020). The most effective way to manage plant diseases is to use healthy,
disease-free plants. To ensure food security and minimize crop losses, continuous
and immediate monitoring and pathogen identification efforts must be undertaken before
planting crops in the field (Kumar et al., 2017; Bhardwaj et al., 2019; Kumar et al.,
2020b; Tiwari et al., 2020; Tiwari et al., 2022; Rahman et al., 2023).
Increasing sensitivity and specificity of disease monitoring in the field have been
enabled by advances in real-time diagnostics. There are several techniques available
for detecting plant pathogens, such as enzyme-linked immunosorbent assay (ELISA),
polymerase chain reaction (PCR), real-time PCR, fluorescence in situ hybridization
(FISH), and flow cytometry. However, these methods often suffer from certain limitations,
including being time-consuming, costly, and requiring highly skilled personnel for
their execution (Kumar et al., 2020a; Kumar et al., 2022). Therefore, plant pathology
is focusing on rapid, accurate, and cost-effective diagnostics, especially for emerging
diseases or elusive pathogens with subtle initial symptoms.
Moreover, diagnostic laboratories have become increasingly dependent on innovative
diagnostic tools designed for field use in an interconnected global environment. The
tools used ensure that instruments and techniques are operationally relevant. Nanotechnology
and biosensor-based diagnostics, along with portable systems integrated with the Internet
of Things (IoT), have revolutionized the field of pathogen detection. These advancements
have led to the development of isothermal amplification-based nucleic acid visual
detection systems, which are highly efficient in identifying pathogens. Key technologies
within this framework include Loop-mediated isothermal amplification (LAMP), rolling
circle amplification (RCA), recombinase polymerase assay (RPA), and CRISPR/Cas (Raigond
et al., 2020; Kumar et al., 2021; Watpade et al., 2023).
The latest advancements in real-time plant pathogen diagnostics
To prevent an outbreak of plant diseases, farmers worldwide must detect them quickly
and accurately. Rapid diagnosis of pathogens is, therefore, necessary to reduce yield
losses. We gathered the latest research on field-level diagnostics for real-time identification
and timely management of plant diseases in crops for this Research Topic. The eleven
research articles on diagnostics cover a broad range of topics, including the diagnosis
and management of bacterial, fungal, viral, phytoplasma, and nonparasitic diseases.
Cheng et al. identified the proteins unaffected by Pst DC3000 infection by mass spectrometry-based
label-free quantification (LFQ) and demonstrated the capability of this to quantify
protein abundance and the possibility of extending protein expression studies to transcripts
in Arabidopsis. Ren et al. tested 100 inter simple sequence repeats (ISSR) primers
and generated a species-specific fragment (515 bp) with ISSR 827 against Tilletia
caries. In addition, they developed a super-sensitive quantitative real-time polymerase
chain reaction (qRT-PCR) with a detection limit of 2.4 fg/μL, and droplet digital
PCR (ddPCR) with a detection limit of 0.24 fg/μL. In a study by Logeshwari et al.,
a LAMP was developed for highly sensitive detection of Sarocladium oryzae at concentrations
as low as 10 fg in 30 minutes at 65°C. LAMP was validated using live infected tissues,
weeds and seeds collected from different locations in Tamil Nadu. Using reverse transcription
recombinase-amplification (RT-RAA) and CRISPR/Cas12a-based lateral flow assays, Lei
et al. developed a rapid detection method that can detect 2.5 copies of the coat protein
gene of MCMV, using 0.96 pg of total RNA extracted from maize leaves infected with
MCMV. To make the method more feasible for field detection, crude virus extraction
of plant RNA combined with one-tube RT-RAA/CRISPR-Cas12a reaction was implemented
on a portable metal incubator (37-42 0C).
Awan et al. conducted antifungal bioassays, and the metabolites extracted from BS-01
exhibited the most potent inhibition of fungal biomass. The extracellular metabolites
displayed an impressive inhibition range of 69-98%, while the intracellular metabolites
showed inhibition ranging from 48% to 85%. In comparison, the metabolites extracted
using n-hexane demonstrated inhibition percentages of 63-88% for extracellular metabolites
and 35-62% for intracellular metabolites. Similarly, the use of dichloromethane resulted
in inhibition percentages of 41-74% for extracellular metabolites and 42-74% for intracellular
metabolites. In growth chamber bioassays, both foliar application and seed application
of BS-01 significantly reduced Alternaria solani load on inoculated tomato foliage.
To improve Plant parasitic nematodes (PPNs) identification and detection, Shao et al.
reviewed the latest research advances and diagnostic approaches and techniques. Morphological
characters alone are not sufficient to identify PPNs because they often have interspecific
overlays and wide intraspecific variations. PPNs can now be diagnosed directly in
the field using newly developed isothermal amplification technologies and remote sensing
methods. Lal et al. studied the worldwide research on real-time PCR-based pathogen
detection from 2001 to 2021 that was used for any diagnostic assay or gene expression
level study. According to the analysis, research on RT-PCR-based pathogen detection
is booming and should be strengthened by using modern diagnostic tools and collaboration
among labs equipped with the necessary equipment. Using crude sap lysed in 0.5M NaOH
solution as a template and purified DNA/cDNA as a primer, Kishan et al. developed
an isothermal-based recombinase polymerase amplification (RPA) method for the detection
of Grapevine geminivirus A (GGVA) in grapevine samples. This assay has the advantage
of not requiring purification or isolation of viral DNA and can be performed at a
wide range of temperatures (18-46°C) for 10-40 minutes, making it an effective and
rapid way to detect grapevine GGVA. Buttar et al. demonstrated that three applications
of Trifloxystrobin+ Tebuconazole 75% WG @ 0.07% were the most effective against pod
rot disease on two mungbean cultivars, ML 2056 and SML 668. ML 2524, among the tested
genotypes, exhibited resistance to pod rot disease, with an incidence of 15.62% and
a severity of 7.69%. A new protocol was developed by Moran et al. that does not require
nucleic acid purification or specialized equipment, making it ideal for field use.
Primer and probe targeting a region of the fusA gene show 94-100% specificity both
in vitro and in silico for the `Ca. Liberibacter´ species associated with HLB. HLB-infected
plant and insect material can be detected with a reliable limit of 101 copies per
microliter using the new protocol. Chauhan et al. studied biochemical mechanisms associated
with cotton leaf curl disease (CLCuD) resistance. High-diseased plants of the susceptible
hybrid HS 6 had a value of 0.7 mg g-1 at 60 DAS. At 90 DAS, resistant cultivars exhibited
the highest phenol content (0.70 mg g-1). HS 6 (9.4 mg g-1) and RCH 134 BG-II (10.5
mg g-1) showed the lowest protein activity at 120 DAS. CLCuV protection in cotton
begins with protein activity, one of the primary biochemical compounds. In cotton,
phenol and tannin are the secondary levels of defense, showing significant increases
in their levels while imparting resistance against CLCuV.
Conclusions and perspectives
In conclusion, the Research Topic addresses the critical need for early detection
and accurate diagnosis of plant pathogens to mitigate crop losses and ensure food
security. It emphasizes the development of diagnostic techniques and tools that are
simple, specific, rapid, and cost-effective. The focus was on advancing field-deployable
molecular diagnostics that enable on-the-spot pathogen detection and immediate response.
Isothermal amplification techniques such as RPA have gained prominence in field-deployable
diagnostics. This technique amplifies targeted nucleic acids of pathogens at a constant
temperature, eliminating the need for complex thermal cycling equipment. They are
simple, fast, and robust, making them suitable for on-site detection even in resource-limited
settings. CRISPR/Cas technologies have also shown promise in plant pathogen detection.
These methods leverage the Cas enzyme’s ability to target and cleave specific sequences
in the pathogen’s DNA or RNA. Coupled with a detection system, CRISPR-based diagnostics
enable rapid identification of pathogens in the field, facilitating real-time disease
monitoring and control. Overall, these advancements in field-deployable molecular
diagnostics, including portable systems interconnected with isothermal amplification
techniques like LAMP and RPA, and CRISPR/Cas technologies, are revolutionizing the
field of plant pathology. They provide rapid, reliable, and on-the-spot pathogen detection
capabilities, empowering farmers, researchers, and agricultural professionals to make
informed decisions and take immediate action to protect crops from the devastating
effects of plant pathogens.
Author contributions
RK: Conceptualization, Resources, Visualization, Writing –original draft, Writing
– review & editing, MKL: Conceptualization, Resources, Visualization, Writing – original
draft, Writing – review & editing, PP: Conceptualization, Resources, Visualization,
Writing – original draft, Writing – review & editing. RKT: Data curation, Formal Analysis,
Investigation, Methodology, Resources, Software, Writing – original draft, Writing
– review & editing.