Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation

Background Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. Results A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) ‘pulses’ of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). Conclusions Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease.

Citrus Huanglongbing Detection Based on Multi-Modal Feature Fusion Learning

Citrus Huanglongbing (HLB), also named citrus greening disease, occurs worldwide and is known as a citrus cancer without an effective treatment. The symptoms of HLB are similar to those of nutritional deficiency or other disease. The methods based on single-source information, such as RGB images or hyperspectral data, are not able to achieve great detection performance. In this study, a multi-modal feature fusion network, combining a RGB image network and hyperspectral band extraction network, was proposed to recognize HLB from four categories (HLB, suspected HLB, Zn-deficient, and healthy). Three contributions including a dimension-reduction scheme for hyperspectral data based on a soft attention mechanism, a feature fusion proposal based on a bilinear fusion method, and auxiliary classifiers to extract more useful information are introduced in this manuscript. The multi-modal feature fusion network can effectively classify the above four types of citrus leaves and is better than single-modal classifiers. In experiments, the highest accuracy of multi-modal network recognition was 97.89% when the amount of data was not very abundant (1,325 images of the four aforementioned types and 1,325 pieces of hyperspectral data), while the single-modal network with RGB images only achieved 87.98% recognition and the single-modal network using hyperspectral information only 89%. Results show that the proposed multi-modal network implementing the concept of multi-source information fusion provides a better way to detect citrus HLB and citrus deficiency.

Genome-Wide Identification and Functional Characterization of GATA Transcription Factor Gene Family in Alternaria alternata

In the present study, we identified six GATA transcription factors (AaAreA, AaAreB, AaLreA, AaLreB, AaNsdD, and AaSreA) and characterized their functions in response to environmental stress and virulence in the tangerine pathotype of Alternaria alternata. The targeted gene knockout of each of the GATA-coding genes decreased the growth to varying degrees. The mutation of AaAreA, AaAreB, AaLreB, or AaNsdD decreased the conidiation. All the GATA transcription factors were found to be required for tolerance to cumyl hydroperoxide and tert-butyl-hydroperoxide (oxidants) and Congo red (a cell-wall-destructing agent). Pathogenicity assays assessed on detached citrus leaves revealed that mutations of AaAreA, AaLreA, AaLreB, or AaNsdD significantly decreased the fungal virulence. A comparative transcriptome analysis between the ∆AreA mutant and the wild-type strain revealed that the inactivation of AaAreA led to alterations in the expression of genes involved in a number of biological processes, including oxidoreductase activity, amino acid metabolism, and secondary metabolite biogenesis. Taken together, our findings revealed that GATA-coding genes play diverse roles in response to environmental stress and are important regulators involved in fungal development, conidiation, ROS detoxification, as well as pathogenesis. This study, for the first time, systemically underlines the critical role of GATA transcription factors in response to environmental stress and virulence in A. alternata.

Parasitoid vectors a plant pathogen, potentially diminishing the benefits it confers as a biological control agent

Huanglongbing (HLB) is a destructive disease of citrus primarily transmitted by the Asian citrus psyllid (ACP). Biocontrol of ACP is an environmentally sustainable alternative to chemicals. However, the risk of parasitoid rational application in ACP biocontrol has never been evaluated. Here we show, the dominant parasitoid of ACP, Tamarixia radiata, can acquire the HLB pathogen Candidatus Liberibacter asiaticus (CLas) and transmit it horizontally when probing ACP nymphs. If these ACP nymphs survive the probing, develop to adults and move to healthy plants, CLas can be transmitted to citrus leaves during feeding. We illustrate the formerly unrecognized risk that a parasitoid can potentially serve as a phoretic vector of the pathogen transmitted by its host, thus potentially diminishing some of the benefits it confers via biocontrol. Our findings present a significant caution to the strategy of using parasitoids in orchards with different infection status of insect-vectored pathogens.

Influence of Rootstock on the Leaf Volatile Organic Compounds of Citrus Scion Is More Pronounced after the Infestation with Diaphorina citri

Nowadays, citrus greening or Huanglongbing is considered the most destructive disease in the citrus industry worldwide. In the Americas and Asia, the disease is caused by the putative pathogen, ‘Candidatus Liberibacter asiaticus’ and transmitted by the psyllid vector, Diaphorina citri. It has been shown that volatile organic compounds (VOC) that are released from citrus leaves attract the psyllid vector. Herein, we tested whether the rootstock influenced the stored VOC profile in the scion leaves and if these influences were altered after infestation with D. citri. The VOC profiles of the hexane-extracted leaves of the mandarin hybrid ‘Sugar Belle’ that were grafted on three different rootstocks (C-35, sour orange (SO), and US-897) with and without infestation with D. citri were studied. The GC-MS analysis showed that the scion VOC profiles of the non-infested control trees were similar to each other, and rootstock was not a strong influence. However, after one month of infestation with D. citri, clear differences in the scion VOC profiles appeared that were rootstock dependent. Although the total scion leaf VOC content did not differ between the three rootstocks, the infestation increased scion monoterpenes significantly on US-897 and C-35 rootstock, increased terpene alcohols on US-897 and SO rootstock, and increased sesquiterpenes on SO. Infestation with D. citri significantly reduced fatty acids and fatty acid esters across all of the rootstocks. Therefore, our results suggest that rootstock choice could influence scions with an inducible volatile defense by enhancing the amounts of VOCs that are available for repelling vectors or for signaling to their natural enemies or parasitoids. According to this study, US-897 may be the best choice among the three that were studied herein, due to its diverse and robust VOC defense response to infestation with D. citri.

Crude Extracts and Alkaloids Derived from Ipomoea-Periglandula Symbiotic Association Cause Mortality of Asian Citrus Psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae)

Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is an important economic pest of citrus crops because it vectors the causal pathogen of huanglongbing (HLB; aka citrus greening). Population suppression of D. citri with insecticides has been disproportionally relied on for HLB management and a greater diversity of more sustainable tools is needed. Periglandula spp. is a fungal endosymbiont (family Clavicipitaceae) that forms a mutualistic relationship with members of plants in family Convolvulaceae. This association results in the production of ergot alkaloids that were previously documented as having psyllicidal properties. We investigated the mortality and behavior of D. citri exposed to crude extracts from morning glories in the plant family Convolvulaceae, as well as synthetic ergot alkaloids. Nymphs and adults were exposed to the crude plant extracts from Periglandula positive species of Convolvulaceae, as well as five synthetic ergot alkaloids. Treatments were prepared by exposing clippings of citrus to 100 ng/µL of crude extract from Periglandula-positive species of Ipomoea (I. imperati, I. leptophylla, I. pandurata and I. tricolor), and Turbina corymbosa, and from one Periglandula-negative species (I. alba) (100 ng/µL). Mortality of adult and nymphal D. citri was significantly higher than the control after exposure to extracts from I. tricolor and I. imperati. The synthetic ergot alkaloids, lysergol (10–100 ng/µL), ergonovine maleate (100 ng/µL), agroclavine (10–100 ng/µL), and ergosine (10–100 ng/µL) increased mortality of D. citri nymphs, while ergosine (100 ng/µL) and agroclavine (100 ng/µL) increased mortality of adults compared to water controls. Fewer D. citri adults settled on plants treated with crude extracts or synthetic ergot alkaloids than on water controls at 48 h after release. D. citri that fed on citrus leaves treated with 10 ng/μL solution of crude extract from the Periglandula-positive species Ipomoea (I. imperati, I. leptophylla, I. pandurata, I. tricolor), and Turbina corymbosa excreted significantly less honeydew compared with a negative water control and extract from Periglandula-negative species (I. alba). Our results indicate that crude extracts and ergot alkaloids exhibit toxic and sub-lethal effects on D. citri that could be useful for management of this pest.

Effect of Larvicidal Extract N-Hexane Lime Leaves (Citrus hystrix) on Larva Instar III Mosquito (Culex quinquefasciatus)

Culex quinquefasciatus mosquitoes are a vector of transmission of several types of diseases, such as filariasis, Japanese encephalitis, and dirofilariasis. Larval control is the key strategy of disease control programs caused by vectors around the world because it can prevent larvae to enter the adult stage. Control of mosquito larvae that are often used is chemically controlled. Citrus hystrix is a natural plant and contains chemical compounds that have biological activity, such as flavonoids, carotenoids, and limonoids. The present study aimed to determine the effect of citrus leaf extract (Citrus hystrix) on the mortality of the larva Culex quinquefasciatus. The method used in the current study was a laboratory experimental study method with the experimental design using a completely randomized design. The research plot carried out was the rearing larvae of Culex quinquefasciatus, the manufacture of citrus leaf extract, the manufacture of larvicidal material, and the observation phase after treatment. Extract N-hexane omplet name hystrix leaf has high toxicity because it contains essential oils, flavonoids, alkaloids, terpenoids, saponins, and Limonoids. The Optimal concentration of N-hexane extracts of citrus leaves that lead to larval mortality of Culex quinquefasciatus in vitro amounted to 93.33% on 4 hours of observation with a concentration of 4000 ppm. Extract N-hexane lime leaf has a larvicidal effect on the larva Culex quinquefasciatus in vitro.

Struktur Sel Sekresi Daun Jeruk Kalamansi (Citrus microcarpa Bunge.) di Pulau Ambon

Perbedaan lokasi tumbuh dapat mengakibatkan perbedaan penampilan fenotipik tanaman yang dapat diamati secara morfologi dan anatomi seperti struktur anatomi sel sekretori. Tujuan penelitian ini adalah untuk mengetahui struktur sel sekretori daun jeruk kalamansi di pulau Ambon. Metode jelajah dilakukan pada 13 lokasi di Pulau Ambon untuk koleksi sampel, dan pada setiap lokasi diambil 3 tanaman sebagai 3 ulangan. Tiap tanaman diambil 5 daun pada setiap sisi pohon tanaman jeruk kalamansi dengan ukuran panjang 5-7 cm dan warna hijau tua. Pembuatan preparat mengikuti metode free hand section. Pengamatan menggunakan kamera Optilab pada mikroskop Olympus dengan perbesaran 400x. Pengukuran diameter sel menggunakan fitur measure pada software Image Ruster. Data kualitatif berupa struktur sel sekresi daun jeruk Kalamansi ditampilkan dalam bentuk gambar dan dideskripsikan sesuai hasil yang terlihat, sedangkan data hasil pengukuran diameter sel sekresi adalah rerata 3 ulangan dan ditampilkan sebagai mean ± standar deviasi (SD). Hasil penelitian menunjukkan adanya sel sekretori yang berjumlah satu sel. Struktur sel sekretori terdiri dari sel epitel, sel selubung, dan rongga sekretori. Bentuk sel sekresi ada yang bulat dan lonjong. Diameter rongga sekretori berkisar antara 106,08-167,60 µm. Berdasarkan hasil penelitian, maka dapat disimpulkan bahwa sel sekresi pada daun jeruk kalamansi pada lokasi-lokasi berbeda di Pulau Ambon bervariasi baik bentuk maupun ukurannya. Differences in habitat can induce differences in the phenotypic appearance of plants that can be observed morphologically and anatomically such as the anatomical structure of secretory cells. The purpose of this study was to determine the structure of the secretory cells in the leaves of Calamansy citrus in Ambon island. Tracking method was done for sample collections, and at each location 3 plants were taken as replicates. Each plant was taken 5 leaves with a length of 5-7 cm and dark green color. Prior to be observed, the fresh sample was done with free-hand section method. Microscopy observations were done by a light microscope at 400x magnification. Measurement of cell diameter was done by the measure feature in Image Ruster software. Qualitative data such as secretory cell structures of Calamansy citrus leaves were shown in form of images and described according to the results, while the data of the measurement of secretory cell diameters is the average of 3 replications and was shown as mean ± standard deviation (SD). The results showed the presence of secretory cells which amounted to one cell. The secretory cell structure is composed of epithelial cells, sheath cells, and secretory cavities. Cell shapes vary, including round and oval. The diameter of the secretory cell cavity ranges from 106.08-167.60 µm.

USING HISTORIC SITES IN DETERMINING PLANT-SOIL INTERACTIONS UNDER LONG TERM CONTAMINATED SOILS

The relationships between heavy metals in soils and crop tissues are critical in understanding the potential risk to crop yields from Zn, Cu and Ni (the principal phytotoxic elements of concern in sludge), and the possible effects on dietary Cd (the critical zootoxic element that is labile in sludge-treated soil and readily absorbed by plants. These relationships cannot be derived from the agronomic trials because the quantities of heavy metals applied to the soil in sludge are small. These are field sites have been treated with sludge for many years in operational practice and where the oncentrations of heavy metals have been significantly raised above background values, representing potentially a worse-case of soil contamination. The purpose of the study was to provide a surrogate for long-term sludge-treated agricultural soil by examining the effects on crops of heavy metals in soil irrigated with raw sewage effluent for periods of more than 80 years and containing significantly elevated concentrations of heavy metals. Methodology: Two surveys of the Gabal El Asfar Old Farm were undertaken to assess the long-term effects of heavy metals in sludge-treated soil on crop quality. In the first survey, the relationships between total and DTPA extractable heavy metals in soil and concentrations in citrus fruit were examined. Concentrations of heavy metals in leaves of citrus were measured in the second survey and related to total and DTPA extractable metals in soil. The heavy metal contents of citrus leaves and fruit (orange - eleven sampling sites; mandarin - four sampling sites), and total and DTPA extractable concentrations in soils were measured in samples collected from different areas of the Farm during two site surveys. Total and DTPA concentrations of heavy metals in the surveyed soils showed significant enrichment by long-term irrigation with sewage effluent. For example, the maximum total concentrations of Zn and Cu were 530 and 366 mg kg-1, respectively, representing a potential risk to crop yields The maximum Cd concentration detected was 9 mg kg-1 and may be a potential risk to the human food chain from uptake into staple crops grown at the farm. DTPA extractable metals were significantly (P<0.001) correlated with the total contents of Zn (r=0.91***), Cu (r=0.83***), Ni (r=0.63***) and Pb (r=0.85***) in soil when data from both surveys were pooled for statistical evaluation. There was also evidence of a weak relationship between DTPA extractable Cd and the total soil Cadmium is the only element of concern in terms of the risk to human health from uptake into food crops grown on sludge-treated soil. The total Cd concentration in soil was raised to a value 3 times the maximum EU limit for this element in sludge-treated agricultural soil. Despite the marked increase in soil Cd content, there was no detectable transfer into citrus leaves or fruit (Figure 1). The absence of Cd uptake into citrus fruit is to be expected because fruits are amongst the least sensitive plant parts to Cd accumulation. These data emphasise the minimal risk to the human diet from Cd in fruit crops grown on sludge-treated soil. . In all cases, leaf tissue concentrations were low and in some cases Cu status was below the deficiency threshold. The Cd content in leaves was small and generally <0.02 mg kg-1 DM.