Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

Immune Cell Degranulation in Fungal Host Defence.

Humans have developed complex immune systems that defend against invading microbes, including fungal pathogens. Many highly specialized cells of the immune system share the ability to store antimicrobial compounds in membrane bound organelles that can be immediately deployed to eradicate or inhibit growth of invading pathogens. These membrane-bound organelles consist of secretory vesicles or granules, which move to the surface of the cell, where they fuse with the plasma membrane to release their contents in the process of degranulation. Lymphocytes, macrophages, neutrophils, mast cells, eosinophils, and basophils all degranulate in fungal host defence. While anti-microbial secretory vesicles are shared among different immune cell types, information about each cell type has emerged independently leading to an uncoordinated and confusing classification of granules and incomplete description of the mechanism by which they are deployed. While there are important differences, there are many similarities in granule morphology, granule content, stimulus for degranulation, granule trafficking, and release of granules against fungal pathogens. In this review, we describe the similarities and differences in an attempt to translate knowledge from one immune cell to another that may facilitate further studies in the context of fungal host defence.

Phagosomal F-Actin Retention by Cryptococcus gattii Induces Dendritic Cell Immunoparalysis.

Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity.IMPORTANCECryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii, which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.

Beta3-Tubulin is Critical for Microtubule Dynamics, Cell Cycle Regulation, and Spontaneous Release of Microvesicles in Human Malignant Melanoma Cells (A375).

Microtubules (MTs), microfilaments, and intermediate filaments, the main constituents of the cytoskeleton, undergo continuous structural changes (metamorphosis), which are central to cellular growth, division, and release of microvesicles (MVs). Altered MTs dynamics, uncontrolled proliferation, and increased production of MVs are hallmarks of carcinogenesis. Class III beta-tubulin (ß3-tubulin), one of seven ß-tubulin isotypes, is a primary component of MT, which correlates with enhanced neoplastic cell survival, metastasis and resistance to chemotherapy. We studied the effects of ß3-tubulin gene silencing on MTs dynamics, cell cycle, and MVs release in human malignant melanoma cells (A375). The knockdown of ß3-tubulin induced G2/M cell cycle arrest, impaired MTs dynamics, and reduced spontaneous MVs release. Additional studies are therefore required to elucidate the pathophysiologic and therapeutic role of ß3-tubulin in melanoma.

Natural killer cells kill Burkholderia cepacia complex via a contact-dependent and cytolytic mechanism.

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.

Microbial killing by NK cells.

It is now evident that NK cells kill bacteria, fungi, and parasites in addition to tumor and virus-infected cells. In addition to a number of recent publications that have identified the receptors and ligands, and mechanisms of cytotoxicity, new insights are reflected in the reports from researchers all over the world at the 17th Meeting of the Society for Natural Immunity held in San Antonio, TX, USA from May 28 through June 1, 2018. We will provide an overview of the field and discuss how the presentations at the meeting might shape our knowledge and future directions in the field.

Granule-Dependent NK Cell Killing of Cryptococcus Requires Kinesin to Reposition the Cytolytic Machinery for Directed Cytotoxicity.

Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing.

Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida.

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that ß-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that ß-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, ß-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble ß-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating ß-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

NKp46 Is an NK Cell Fungicidal Pattern Recognition Receptor.

Natural killer (NK) cells are an important contributor to innate host defense because of their role in direct microbial recognition and killing. Vitenshtein et al. make an important contribution by demonstrating that NK cells kill Candida glabrata using the NK activating receptor, NKp46, which recognizes the Epa adhesins.

Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans.

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.

Cryptococcus gattii Capsule Blocks Surface Recognition Required for Dendritic Cell Maturation Independent of Internalization and Antigen Processing.

Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation.

Insulin and hypoxia-inducible factor-1 cooperate in pancreatic cancer cells to increase cell viability.

The aim of the present study was to investigate whether interstitial insulin and cancer-induced hypoxia-inducible factor-1 (HIF-1) cooperate in pancreatic cancer cells. A population of 45 nude mice were divided into one intact control group and six pancreatic tumor-carrier groups. Pancreatic tumors were generated using HIF-1-positive wild-type MiaPaCa2 (wt-MiaPaCa2) pancreatic cancer cells in three groups of carriers and MiaPaCa2 cells transfected with small interfering RNA against HIF-1α (si-MiaPaCa2 cells) in the other three carrier groups. To vary the intrapancreatic insulin levels, tumor-carrying mice were subjected to one of the following conditions: i) Untreated, ii) single injection of the ß-cell toxin streptozotosin prior to cancer cell transplantation and iii) daily injection of insulin following cancer cell transplantation. After 12 weeks, tumor viability was assessed by histological analysis. Western blotting of the tumor grafts was performed to determine the protein expression levels of insulin receptor (IR) and two downstream proteins, hexokinase-II (HK-II) and vascular endothelial growth factor (VEGF). Histologically, the greatest viability was observed in wt-MiaPaCa2 tumors with carriers that remained untreated. These tumors also exhibited greater IR expression than their si-MiaPaCa2 counterparts, indicating that HIF-1 is necessary for basal expression of IR. However, IR expression was increased in wt-MiaPaCa2 and si-MiaPaCa2 tumors when the carriers were treated with exogenous insulin. This indicates that the insulin-induced IR expression was independent of HIF-1. Notably, the insulin-induced IR expression was associated with increased HK-II and VEGF expression in wt-MiaPaCa2 tumors but not si-MiaPaC2 tumors. Therefore, the present study proposes that insulin and HIF-1 may cooperate to increase pancreatic cancer cell viability. Furthermore, the HIF-1 signaling pathway is required for insulin-induced HK-II and VEGF expression, as well as basal IR expression levels in pancreatic cancer cells.

Gene Expressing and sRNA Sequencing Show That Gene Differentiation Associates with a Yellow Acer palmatum Mutant Leaf in Different Light Conditions.

Acer palmatum Thunb., like other maples, is a widely ornamental-use small woody tree for leaf shapes and colors. Interestingly, we found a yellow-leaves mutant "Jingling Huangfeng" turned to green when grown in shade or low-density light condition. In order to study the potential mechanism, we performed high-throughput sequencing and obtained 1,082 DEGs in leaves grown in different light conditions that result in A. palmatum significant morphological and physiological changes. A total of 989 DEGs were annotated and clustered, of which many DEGs were found associating with the photosynthesis activity and pigment synthesis. The expression of CHS and FDR gene was higher while the expression of FLS gene was lower in full-sunlight condition; this may cause more colorful substance like chalcone and anthocyanin that were produced in full-light condition, thus turning the foliage to yellow. Moreover, this is the first available miRNA collection which contains 67 miRNAs of A. palmatum, including 46 conserved miRNAs and 21 novel miRNAs. To get better understanding of which pathways these miRNAs involved, 102 Unigenes were found to be potential targets of them. These results will provide valuable genetic resources for further study on the molecular mechanisms of Acer palmatum leaf coloration.

The NK receptor NKp30 mediates direct fungal recognition and killing and is diminished in NK cells from HIV-infected patients.

Natural killer (NK) cells are a subset of immune effectors that directly bind and kill fungi via a perforin-dependent mechanism. The receptor mediating this activity and its potential role in disease remain unknown. Using an unbiased approach, we determined that NKp30 is responsible for recognition and killing of the fungal pathogens Cryptococcus and Candida. NKp30 was required for NK cell-fungal conjugate formation, phosphatidylinositol 3-kinase (PI3K) signaling, and perforin release. Because fungal infections are a leading cause of death in AIDS patients, we examined NKp30 expression in HIV-infected patients. NK cells from these patients had diminished NKp30 expression, defective perforin release, and blunted microbicidal activity. Surprisingly, interleukin-12 (IL-12) restored NKp30 expression and fungal killing. Thus, the NKp30 receptor plays a critical role in NK cell antifungal cytotoxicity, and diminished expression of NKp30 is responsible for defective antifungal activity of NK cells from HIV-infected patients, which can be corrected with IL-12.

Requirement and redundancy of the Src family kinases Fyn and Lyn in perforin-dependent killing of Cryptococcus neoformans by NK cells.

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.

An acidic microenvironment increases NK cell killing of Cryptococcus neoformans and Cryptococcus gattii by enhancing perforin degranulation.

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Cryptococcus gattii is killed by dendritic cells, but evades adaptive immunity by failing to induce dendritic cell maturation.

During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.

Cryptococcus.

Cryptococcus is a unique environmental fungus. Among the more than three dozen species of Cryptococcus, only C. neoformans and C. gattii commonly cause disease. Although many of these infections occur in immunocompromised patients, C. gattii has recently come to public attention because of an outbreak of devastating illness in immunocompetent individuals. The polysaccharide capsule of Cryptococcus is a major virulence factor, and in addition to surrounding the organism, it is also released into the environment. Cryptococcus is believed to enter the body through the lung causing pulmonary disease, but because of its neurotropic nature, the central nervous system is a major target organ. The major risk factors include HIV and organ transplantation. Depending on the site of infection and the patient's immune status, the clinical manifestations vary from asymptomatic to severe life-threatening disease. Treatment regimens depend on the immune status of the patient and the severity of the disease, and include both polyene and imidazole antifungal agents in addition to surgical adjuvant therapy. However, despite antifungal therapy, the mortality remains between 10 and 25% in patients with AIDS, and at least one-third of patients with cryptococcal meningitis experience mycological or clinical failure. Consequently, the mechanism of cryptococcal invasion, immune response, pathogenesis, and treatment continue to be areas of active study. With our advancing knowledge in these areas, we aim at better management for this devastating group of infections.

Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain.

Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection.

[Effects of labor-saving rice cultivation modes on the diversity of potential weed communities in paddy fields].

Aimed to understand the effects of various labor-saving rice cultivation modes on the diversity of potential weed communities in paddy fields, an investigation was made on the quantitative characteristics of the weed seed bank under dry direct seeding, water direct seeding, seedling throwing, mechanized-transplanting, wheat-rice interplanting, and conventional manual transplanting. Under dry direct seeding, the density of the weed seed bank was up to 228,416 seeds x m(-2), being significantly higher than that under the other five cultivation modes. Wheat-rice interplanting ranked the second place. The seed density of sedge weeds under dry direct seeding and that of broad leaf weeds under wheat-rice interplanting were significantly higher than the seed densities of various kinds of weeds under other cultivation modes. Conventional manual transplanting mode had the highest species richness, with Margalef index being 1.86. The diversity indices, including Shannon-Wiener index, Gini index, and Pielou evenness index under water direct seeding and wheat-rice interplanting were higher than those under other cultivation modes. Comparing with conventional manual transplanting mode, the other five cultivation modes had their own dominant species in the potential weed community, and thereby, different labor-saving rice cultivation modes should be applied by turns to control the potential weed community in paddy fields effectively and persistently.