Characterization and fine mapping of cold-inducible parthenocarpy in cucumber (Cucumis sativus L.).

Cold stress detrimentally influences fruit development, leading to a substantial yield reduction in many fruit-bearing vegetables. Cucumber, a vegetable of subtropical origin, is especially sensitive to cold. Cold-inducible parthenocarpy (CIP) promises fruit yield under cold conditions. Previously, we identified a CIP line EC5 in cucumber, which showed strong parthenocarpy and sustained fruit growth under cold conditions (16°C day/10°C night). However, the candidate gene and genetic mechanism underlying CIP in cucumber remain unknown. In this study, both BSA-seq and conventional QTL mapping strategies were employed on F2 populations to delve into the genetic control of CIP. A single QTL, CIP5.1, was consistently mapped across two winter seasons in 2021 and 2022. Fine mapping delimited the CIP locus into a 38.3kb region on chromosome 5, harboring 8 candidate genes. Among these candidates, CsAGL11 (CsaV3_5G040370) was identified, exhibiting multiple deletions/insertions in the promoter and 5'UTR region. The CsAGL11 gene encodes a MADS-box transcription factor protein, which is homologous to the genes previously recognized as negative regulators in ovule and fruit development of Arabidopsis and tomato. Correspondingly, cold treatment resulted in decreased expression of CsAGL11 during the early developmental stage of the fruit in EC5. A promoter activity assay confirmed promoter polymorphisms leading to weak transcriptional activation of CsAGL11 under cold conditions. This study deepens our understanding of the genetic characteristics of CIP and elucidates the potential role of the CsAGL11 gene in developing cucumber cultivars with enhanced fruiting under cold conditions.

Comparative transcriptome analysis of two pomelo accessions with different parthenocarpic ability provides insight into the molecular mechanisms of parthenocarpy in pomelo (Citrus grandis).

Parthenocarpy is an important way for seedless fruit production in citrus. However, the molecular mechanism(s) of parthenocarpy in pomelo is still unknown. Our initial study found significantly different parthenocarpic abilities in Guanximiyou (G) and Shatianyou (S) pomelo following emasculation, and an endogenous hormone content assay revealed that indole-3-acetic acid (IAA), gibberellic acid (GA3) and zeatin (ZT) jointly promoted fruit expansion and cell division in parthenocarpic pomelo (G pomelo). To unravel the underlying molecular mechanism(s), we conducted the first transcriptome analysis on the two pomelo accessions at these two critical stages: the fruit initiation stage and the rapid expansion stage, in order to identify genes associated with parthenocarpy. This analysis yielded approximately 7.86 Gb of high-quality reads, and the subsequent de novo assembly resulted in the identification of 5,792 DEGs (Differentially Expressed Genes). Among these, a range of transcription factor families such as CgERF, CgC2H2, CgbHLH, CgNAC and CgMYB, along with genes like CgLAX2, CgGH3.6 and CgGH3, emerged as potential candidates contributing to pomelo parthenocarpy, as confirmed by qRT-PCR analysis. The present study provides comprehensive transcriptomic profiles of both parthenocarpic and non-parthenocarpic pomelos, reveals several metabolic pathways linked to parthenocarpy, and highlights the significant role of plant hormones in its regulation. These findings deepen our understanding of the molecular mechanisms underlying parthenocarpy in pomelo.

Morphological Changes to Fruit Development Induced by GA3 Application in Sweet Cherry (Prunus avium L.).

Cherry (Prunus avium) fruits are important sources of vitamins, minerals, and nutrients in the human diet; however, they contain a large stone, making them inconvenient to eat 'on the move' and process. The exogenous application of gibberellic acid (GA3) can induce parthenocarpy in a variety of fruits during development. Here, we showed that the application of GA3 to sweet cherry unpollinated pistils acted as a trigger for fruit set and permitted the normal formation of fruit up to a period of twenty-eight days, indicating that gibberellins are involved in the activation of the cell cycle in the ovary wall cells, leading to fruit initiation. However, after this period, fruit development ceased and developing fruit began to be excised from the branch by 35 days post treatment. This work also showed that additional signals are required for the continued development of fully mature parthenocarpic fruit in sweet cherry.

The hormone regulatory mechanism underlying parthenocarpic fruit formation in tomato.

Parthenocarpic fruits, known for their superior taste and reliable yields in adverse conditions, develop without the need for fertilization or pollination. Exploring the physiological and molecular mechanisms behind parthenocarpic fruit development holds both theoretical and practical significance, making it a crucial area of study. This review examines how plant hormones and MADS-box transcription factors control parthenocarpic fruit formation. It delves into various aspects of plant hormones-including auxin, gibberellic acid, cytokinins, ethylene, and abscisic acid-ranging from external application to biosynthesis, metabolism, signaling pathways, and their interplay in influencing parthenocarpic fruit development. The review also explores the involvement of MADS family gene functions in these processes. Lastly, we highlight existing knowledge gaps and propose directions for future research on parthenocarpy.

Parthenocarpy, a pollination-independent fruit set mechanism to ensure yield stability.

Fruit development is essential for flowering plants' reproduction and a significant food source. Climate change threatens fruit yields due to its impact on pollination and fertilization processes, especially vulnerable to extreme temperatures, insufficient light, and pollinator decline. Parthenocarpy, the development of fruit without fertilization, offers a solution, ensuring yield stability in adverse conditions and enhancing fruit quality. Parthenocarpic fruits not only secure agricultural production but also exhibit improved texture, appearance, and shelf life, making them desirable for food processing and other applications. Recent research unveils the molecular mechanisms behind parthenocarpy, implicating transcription factors (TFs), noncoding RNAs, and phytohormones such as auxin, gibberellin (GA), and cytokinin (CK). Here we review recent findings, construct regulatory models, and identify areas for further research.

Highly efficient CRISPR/Cas9-RNP mediated CaPAD1 editing in protoplasts of three pepper (Capsicum annuum L.) cultivars.

Parthenocarpy, characterized by seedless fruit development without pollination or fertilization, offers the advantage of consistent fruit formation, even under challenging conditions such as high temperatures. It can be induced by regulating auxin homeostasis; PAD1 (PARENTAL ADVICE-1) is an inducer of parthenocarpy in Solanaceae plants. However, precise editing of PAD1 is not well studied in peppers. Here, we report a highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) for CaPAD1 editing in three valuable cultivars of pepper (Capsicum annuum L.): Dempsey, a gene-editable bell pepper; C15, a transformable commercial inbred line; and Younggo 4, a Korean landrace. To achieve the seedless pepper trait under high temperatures caused by unstable climate change, we designed five single guide RNAs (sgRNAs) targeting the CaPAD1 gene. We evaluated the in vitro on-target activity of the RNP complexes in three cultivars. Subsequently, we introduced five CRISPR/Cas9-RNP complexes into protoplasts isolated from three pepper leaves and compared indel frequencies and patterns through targeted deep sequencing analyses. We selected two sgRNAs, sgRNA2 and sgRNA5, which had high in vivo target efficiencies for the CaPAD1 gene across the three cultivars and were validated as potential off-targets in their genomes. These findings are expected to be valuable tools for developing new seedless pepper cultivars through precise molecular breeding of recalcitrant crops in response to climate change.

Jasmonate-insensitive mutant jar1b prevents petal elongation and flower opening coupling with parthenocarpic fruit development in Cucurbita pepo.

Jasmonates are growth regulators that play a key role in flower development, fruit ripening, root growth, and plant defence. The study explores the coordination of floral organ maturation to ensure proper flower opening for pollination and fertilization. A new mutant (jar1b) was discovered, lacking petal elongation and flower opening but showing normal pistil and stamen development, leading to parthenocarpic fruit development. The mutation also enhanced the elongation of roots while reducing the formation of root hairs. BSA sequencing showed that jar1b is a missense mutation in the gene CpJAR1B, which encodes the enzyme that catalyzes the conjugation between JA and the amino acid isoleucine. The loss of function mutation in CpJAR1B produced a deficiency in biologically active (+) -7-iso-jasmonoyl-L-isoleucine (JA-Ile), which was not complemented by the paralogous gene CpJAR1A or any other redundant gene. Exogenous application of methyl jasmonate (MeJA) demonstrated that jar1b is partially insensitive to JA in both flowers and roots. Further experimentation involving the combination of JA-Ile deficient and ethylene-deficient, and ET insensitive mutations in double mutants revealed that CpJAR1B mediated ET action in female petal maturation and flower opening, but JA and ET have independent additive effects as negative regulators of the set and development of squash fruits. CpJAR1B also regulated the aperture of male flowers in an ethylene-independent manner. The root phenotype of jar1b and effects of external MeJA treatments indicated that CpJAR1B has a dual role in root development, inhibiting the elongation of primary and secondary roots, but promoting the formation of root hairs.

Genetic and molecular mechanisms underlying the parthenocarpic fruit mutation in tomato.

Parthenocarpy allows fruit set independently of fertilization. In parthenocarpic-prone tomato genotypes, fruit set can be achieved under pollen-limiting environmental conditions and in sterile mutants. Parthenocarpy is also regarded as a quality-related trait, when seedlessness is associated with positive fruit quality aspects. Among the different sources of genetic parthenocarpy described in tomato, the parthenocarpic fruit (pat) mutation is of particular interest because of its strong expressivity, high fruit set, and enhanced fruit quality. The complexity of the pat "syndrome" associates a strong competence for parthenocarpy with a complex floral phenotype involving stamen and ovule developmental aberrations. To understand the genetic basis of the phenotype, we mapped the pat locus within a 0.19-cM window of Chr3, comprising nine coding loci. A non-tolerated missense mutation found in the 14th exon of Solyc03g120910, the tomato ortholog of the Arabidopsis HD-Zip III transcription factor HB15 (SlHB15), cosegregated with the pat phenotype. The role of SlHB15 in tomato reproductive development was supported by its expression in developing ovules. The link between pat and SlHB15 was validated by complementation and knock out experiments by co-suppression and CRISPR/Cas9 approaches. Comparing the phenotypes of pat and those of Arabidopsis HB15 mutants, we argued that the gene plays similar functions in species with fleshy and dry fruits, supporting a conserved mechanism of fruit set regulation in plants.

Generation of parthenocarpic tomato plants in multiple elite cultivars using the CRISPR/Cas9 system.

Tomato (Solanum lycopersicum L.) is one of the most important crops in the world for its fruit production. Advances in cutting-edge techniques have enabled the development of numerous critical traits related to the quality and quantity of tomatoes. Genetic engineering techniques, such as gene transformation and gene editing, have emerged as powerful tools for generating new plant varieties with superior traits. In this study, we induced parthenocarpic traits in a population of elite tomato (ET) lines. At first, the adaptability of ET lines to genetic transformation was evaluated to identify the best-performing lines by transforming the SlANT1 gene overexpression cassette and then later used to produce the SlIAA9 knockout lines using the CRISPR/Cas9 system. ET5 and ET8 emerged as excellent materials for these techniques and showed higher efficiency. Typical phenotypes of knockout sliaa9 were clearly visible in G0 and G1 plants, in which simple leaves and parthenocarpic fruits were observed. The high efficiency of the CRISPR/Cas9 system in developing new tomato varieties with desired traits in a short period was demonstrated by generating T-DNA-free homozygous sliaa9 knockout plants in the G1 generation. Additionally, a simple artificial fertilization method was successfully applied to recover seed production from parthenocarpic plants, securing the use of these varieties as breeding materials. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01452-1.

Parthenocarpy in Cucurbitaceae: Advances for Economic and Environmental Sustainability.

Parthenocarpy is an important agricultural trait that not only produces seedless fruits, but also increases the rate of the fruit set under adverse environmental conditions. The study of parthenocarpy in Cucurbitaceae crops has considerable implications for cultivar improvement. This article provides a comprehensive review of relevant studies on the parthenocarpic traits of several major Cucurbitaceae crops and offers a perspective on future developments and research directions.

Floral Development on Vitis vinifera Is Associated with MADS-Box Transcription Factors through the Transcriptional Regulation of VviZIP3.

Several grapevine (Vitis vinifera L.) cultivars show a tendency to develop parthenocarpic seedless grapes, affecting fruit yield and quality. This reproductive disorder originates in defective ovule fertilization due to a failure in pollen tube growth. Zinc (Zn) is a crucial trace element, playing a vital role in various physiological and metabolic processes. It is particularly essential for the healthy growth of flowers and fruits. Insufficient zinc has been suggested as a potential reason for issues in this development process. This microelement is taken up through a mechanism that involves transporters, including the ZRT-IRT-like protein (ZIP) gene family, associated with the influx of Zn into the cell. In grapevines, 20 genes for ZIP-type transporters have been described. In this study, we analyzed the expression pattern of VviZIP3 during flower development and employ transgenic methods to assess its transcriptional regulation. Furthermore, through computational examination of the promoter region, we identified two CArG boxes, recognized as responsive elements to MADS transcription factors. These factors play a key role in shaping various components of a flower, such as pollen. Our investigation of the VviZIP3 promoter confirms the functionality of these CArG boxes. Overall, our results suggest that the increased expression of VviZIP3 during flowering is likely under the influence of MADS transcription factors.

MicroRNAs associated with AGL6 and IAA9 function in tomato fruit set.

OBJECTIVE: Fruit set is triggered after ovule fertilization, as a consequence of the downregulation of ovary growth repressors, such as the tomato transcription factors Auxin/indole-3-acetic acid 9 (IAA9) and Agamous-like 6 (AGL6). In a recent work, we developed a method to silence IAA9 and AGL6 in tomato ovaries using exogenous dsRNAs. We also produced small RNA libraries from IAA9- and AGL6-silenced ovaries to confirm the presence of siRNAs, derived from exogenous dsRNA, targeting IAA9 and AGL6. The objective of this work is to exploit these sRNA libraries to identify miRNAs differentially expressed in IAA9- and AGL6-silenced ovaries as compared with unpollinated control ovaries. RESULTS: We identified by RNA sequencing 125 and 104 known and 509 and 516 novel miRNAs from reads mapped to mature or hairpin sequences, respectively. Of the known miRNAs, 7 and 45 were differentially expressed in IAA9- and AGL6-silenced ovaries compared to control ones, respectively. Six miRNAs were common to both datasets, suggesting their importance in the fruit set process. The expression pattern of two of these (miR393 and miR482e-5p) was verified by stem-loop qRT-PCR. The identified miRNAs represent a pool of regulatory sRNAs potentially involved in tomato fruit initiation.

Pollination, pollen tube growth, and fertilization independently contribute to fruit set and development in tomato.

In flowering plants, pollination, pollen tube growth, and fertilization are regarded as the first hierarchical processes of producing offspring. However, their independent contributions to fruit set and development remain unclear. In this study, we examined the effect of three different types of pollen, intact pollen (IP), soft X-ray-treated pollen (XP) and dead pollen (DP), on pollen tube growth, fruit development and gene expression in "Micro-Tom" tomato. Normal germination and pollen tube growth were observed in flowers pollinated with IP; pollen tubes started to penetrate the ovary at 9 h after pollination, and full penetration was achieved after 24 h (IP24h), resulting in ~94% fruit set. At earlier time points (3 and 6 h after pollination; IP3h and IP6h, respectively), pollen tubes were still in the style, and no fruit set was observed. Flowers pollinated with XP followed by style removal after 24 h (XP24h) also demonstrated regular pollen tubes and produced parthenocarpic fruits with ~78% fruit set. As expected, DP could not germinate and failed to activate fruit formation. Histological analysis of the ovary at 2 days after anthesis (DAA) revealed that IP and XP comparably increased cell layers and cell size; however, mature fruits derived from XP were significantly smaller than those derived from IP. Furthermore, there was a high correlation between seed number and fruit size in fruit derived from IP, illustrating the crucial role of fertilization in the latter stages of fruit development. RNA-Seq analysis was carried out in ovaries derived from IP6h, IP24h, XP24h and DP24h in comparison with emasculated and unpollinated ovaries (E) at 2 DAA. The results revealed that 65 genes were differentially expressed (DE) in IP6h ovaries; these genes were closely associated with cell cycle dormancy release pathways. Conversely, 5062 and 4383 DE genes were obtained in IP24h and XP24h ovaries, respectively; top enriched terms were mostly associated with cell division and expansion in addition to the 'plant hormone signal transduction' pathway. These findings indicate that full penetration of pollen tubes can initiate fruit set and development independently of fertilization, most likely by activating the expression of genes regulating cell division and expansion.

Molecular, hormonal, and metabolic mechanisms of fruit set, the ovary-to-fruit transition, in horticultural crops.

Fruit set is the process by which the ovary develops into a fruit and is an important factor in determining fruit yield. Fruit set is induced by two hormones, auxin and gibberellin, and the activation of their signaling pathways, partly by suppressing various negative regulators. Many studies have investigated the structural changes and gene networks in the ovary during fruit set, revealing the cytological and molecular mechanisms. In tomato (Solanum lycopersicum), SlIAA9 and SlDELLA/PROCERA act as auxin and gibberellin signaling repressors, respectively, and are important regulators of the activity of transcription factors and downstream gene expression involved in fruit set. Upon pollination, SlIAA9 and SlDELLA are degraded, which subsequently activates downstream cascades and mainly contributes to active cell division and cell elongation, respectively, in ovaries during fruit setting. According to current knowledge, the gibberellin pathway functions as the most downstream signal in fruit set induction, and therefore its role in fruit set has been extensively explored. Furthermore, multi-omics analysis has revealed the detailed dynamics of gene expression and metabolites downstream of gibberellins, highlighting the rapid activation of central carbon metabolism. This review will outline the relevant mechanisms at the molecular and metabolic levels during fruit set, particularly focusing on tomato.

Parthenocarpy-related genes induced by naphthalene acetic acid in oil palm interspecific O × G [Elaeis oleifera (Kunth) Cortés × Elaeis guineensis Jacq.] hybrids.

Parthenocarpy is the development without fertilization of seedless fruits. In the oil palm industry, the development of parthenocarpic fruits is considered an attractive option to increase palm oil production. Previous studies have shown the application of synthetic auxins in Elaeis guineensis, and interspecific O×G hybrids (Elaeis oleifera (Kunth) Cortés × E. guineensis Jacq.) induces parthenocarpy. The aim of this study was to identify the molecular mechanism through transcriptomics and biology system approach to responding to how the application of NAA induces parthenocarpic fruits in oil palm O×G hybrids. The transcriptome changes were studied in three phenological stages (PS) of the inflorescences: i) PS 603, pre-anthesis III, ii) PS 607, anthesis, and iii) PS 700, fertilized female flower. Each PS was treated with NAA, Pollen, and control (any application). The expression profile was studied at three separate times: five minutes (T0), 24 hours (T1), and 48 h post-treatment (T2). The RNA sequencing (RNA seq) approach was used with 27 oil palm O×G hybrids for a total of 81 raw samples. RNA-Seq showed around 445,920 genes. Numerous differentially expressed genes (DEGs) were involved in pollination, flowering, seed development, hormone biosynthesis, and signal transduction. The expression of the most relevant transcription factors (TF) families was variable and dependent on the stage and time post-treatment. In general, NAA treatment expressed differentially more genes than Pollen. Indeed, the gene co-expression network of Pollen was built with fewer nodes than the NAA treatment. The transcriptional profiles of Auxin-responsive protein and Gibberellin-regulated genes involved in parthenocarpy phenomena agreed with those previously reported in other species. The expression of 13 DEGs was validated by RT-qPCR analysis. This detailed knowledge about the molecular mechanisms involved in parthenocarpy could be used to facilitate the future development of genome editing techniques that enable the production of parthenocarpic O×G hybrid cultivars without growth regulator application.

Transcriptome dataset from Solanum lycopersicum L. cv. Micro-Tom; wild type and two mutants of INDOLE-ACETIC-ACID (SlIAA9) using long-reads sequencing oxford nanopore technologies.

OBJECTIVE: Tomatoes are the most widely consumed fruit vegetable and are relatively easy to cultivate. However, an increase in temperature causes some plants to respond with a decrease in fruit production. So, it is necessary to develop plants resistant to extreme temperature changes. The tomato cv. Micro-Tom has genetic variations in the gene of INDOLE-ACETIC-ACID, namely SlIAA9-3 and SlIAA9-5. However, the genetic information regarding the full-length transcript of the gene from this type of tomato plant is unknown. Therefore, this study aimed to determine the full-length transcript of the genes of these three types of tomatoes using long-reads sequencing technology from Oxford Nanopore. DATA DESCRIPTION: The total RNA from three types of Micro-Tom was isolated with the RNeasy PowerPlant Kit. Then, the RNA sequencing process used PCR-cDNA Barcoding kit - SQK-PCB109 and continued with the processing of raw reads based on the protocol from microbepore protocol ( https://github.com/felixgrunberger/microbepore ). The resulting raw reads were 578 374, 409 905, and 851 948 for wildtype, iaa9-3, and iaa9-5, respectively. After obtaining cleaned reads, each sample was mapped to the tomato reference genome (S. lycopersicum ITAG4.0) with the Minimap2 program. In particular, 965 genes were expressed only in the iaa9-3 mutant, and 2332 genes were expressed only in the iaa9-5 mutant. Whereas in the wild type, 1536 genes are specifically expressed. In cluster analysis using the heatmap analysis, separate groups were obtained between the wild type and the two mutants. This proves an overall difference in transcript levels between the wild type and the mutants.

Seedlessness Trait and Genome Editing-A Review.

Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements.

Jasmonate-deficient mutant lox3a reveals crosstalk between jasmonate and ethylene in the differential regulation of male and female flower opening and early fruit development in Cucurbita pepo.

Jasmonate (JA) has been found to be a relevant hormone in floral development in numerous species, but its function in cucurbit floral development and sex determination is unknown. Crosstalk between JA and ethylene (ET) in the differential regulation of male and female floral development was investigated by using the novel JA-deficient mutant lox3a, and the ET-deficient and -insensitive mutants, aco1a and etr2b, respectively, of Cucurbita pepo. The lox3a mutation suppresses male and female flower opening and induces the development of parthenocarpic fruit. A bulked-segregant analysis coupled with whole genome sequencing and fine mapping approach allowed the identification of lox3a mutation in CpLOX3A, a LIPOXYGENASE gene involved in JA biosynthesis. The reduced JA content and expression of JA-signalling genes in male and female flowers of lox3a, and the rescue of lox3a phenotype by external application of methyl jasmonate (MeJA), demonstrated that JA controls petal elongation and flower opening, as well as fruit abortion in the absence of fertilization. JA also rescued the phenotype of ET mutants aco1a and etr2b, which are both specifically defective in female flower opening and fruit abortion. ET, the sex determining hormone of cucurbits, is induced in female flowers towards anthesis, activating JA production and promoting the aperture of the female flower, and the abortion of the unfertilized ovary. Given the close association between flower closure and parthenocarpic fruit development, we propose that flower opening can act as a switch that triggers fruit set and development in fertilized ovaries, but may alternatively induce the abortion of the unfertilized ovary. Both ET and JA from mature and senescent petals can serve as remote signals that determine the alternative development of the ovary and fruit.

Artificial pollination and use of growth regulators in atemoya 'Gefner' fruits

The objective was to study the effectiveness of the growth regulator (ANA + GA3) associated or not to the application of adjuvant and artificial pollination in 'Gefner' atemoya. The experiment was conducted in the experimental orchard at Florida's Tropical Research and Education Center. The experimental design was in a randomized block, with 14 treatments, 10 repetitions and 3 flowers per plot. The highest percentages of fixed fruits were obtained with hand pollination ­ HP and 450 NAA + 1250 GA3 mg L-1 + adjuvant and HP. The use of hand pollination for 'Gefner' atemoya tree proved to be the most efficient method so far. Applying growth regulator without artificial pollination produces parthenocarpic fruits, however with high rate of abortions, and small fruits. Growth regulators together with hand pollination produces small and uneven fruits, and cause reduction in the fruits' titratable acidity. The use of adjuvant caused low fixation and toxicity to fruits, and its use is not recommended.