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Penthiopyrad Analysis Essay

2.1. Chemical Synthesis

Thirty-two title compounds were prepared according to the methods presented in Scheme 1, including twenty-six 1,3,4-substituted-5-aminopyrazole derivatives, two 1,3,4-substituted pyrazole amides, and four 1,3,4-substituted pyrazole isothiocyanates. Compounds 3ad were obtained by the treatment of the dithioacetal dipotassium salts 2ab with dimethyl sulfate or benzyl chloride in the presence of methanol and H2O. The resulting compounds were then reacted with corresponding hydrazines to afford 1,3,4-substituted-5-aminopyrazole derivatives 126.

Scheme 1. Synthetic route and chemical structures of compounds 132.

Scheme 1. Synthetic route and chemical structures of compounds 132.

In order to explore the effects of substituted pyrazole compounds against pathogenic fungi, amide groups and isothiocyanate groups were introduced. The substituted pyrazole amides 27 and 28 were synthesized by treating aromatic acid with compounds 3 and 4 in the presence of dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP). Preparation of the target 1,3,4-substituted pyrazole isothiocyanate compounds was performed as follows: first, the compounds were converted into 1,3,4-substituted-5-N-(3-bromopropyl)pyrazoles through alkylation using 1,3-dibromopropane. Then, 4ad were reacted with sodium azide to obtain hydrazoate compounds 5ad. Treatment of 5ad with triphenylphosphine and carbon disulfide in THF afforded 2932.

Most of target compounds were synthesized in good yields of 90% or higher and were characterized by 1H-NMR, 13C-NMR and HR-MS (ESI). All spectral and analytical data were consistent with the assigned structures.

2.2. Fungicidal Activity

The initial screening data results for the fungicidal activities of the tested compounds against Botrytis cinerea, Rhizoctonia solani Kuhn, Valsa mali Miyabe et Yamada, Thanatephorus cucumeris (Frank) Donk, Fusarium oxysporum (S-chl) f.sp. cucumerinum Owen, and Fusarium graminearum Schw at a concentration of 100 mg/L are listed in Table 1. Many of the synthesized compounds possessed moderate to high fungicidal activities. Thirteen compounds (24, 6, 8, 10, 2632) showed inhibition rates exceeding 80% against B. cinerea, thirteen compounds (24, 6, 8, 10, 2632) displayed more than 90% inhibition activities against R. solani, fifteen compounds (24, 610, 2632) exhibited over 80% inhibition activities against V. mali, thirteen compounds (24, 6, 8, 10, 2632) possessed over 80% inhibition activities against T. cucumeris, ten compounds (3, 6, 10, 2632) showed more than 80% effects against F. oxysporum, and eleven compounds (2, 3, 6, 10, 2632) displayed more than 90% inhibition activity against F. graminearum. Among these compounds, compounds 3, 6, 2632 showed broad spectra fungicidal activities against all tested phytopathogens with over 90% inhibition rates, especially, compounds 26, 27, 28, 30, 31 which showed 100% activities at a dosage of 100 mg/L. Compound 9 showed special activity against V. mali, displaying 84.30% inhibition activity, and showed low level of activities to B. cinerea, F. oxysporum, and F. graminearum.

Compounds 14, 611, 2632 were found to display good fungicidal activities and were chosen to do a rescreening. The bioassay data is summarized in Table 2. These compounds displayed growth-inhibitory activities with EC50 values ranges from 2.432–10.627, 2.182–11.024, 1.787–12.877, 1.638–10.253, 8.073–15.320, and 6.043–19.701 μg/mL against B. cinerea, R. solani, V. mali, T. cucumeris, F. oxysporum, and F. graminearum, respectively. Among them, compound 26 exhibited the highest activities, with EC50 values of 2.432, 2.182, 1.787, 1.638, 6.986, and 6.043 μg/mL against B. cinerea, R. solani, V. mali, T. cucumeris, F. oxysporum, and F. graminearum, respectively.

Based on the activities of pyrazole derivatives, structure-activity relationships can be discussed. The activities of the target compounds is attributed to the cyano group. Comparing the bioactivity between compounds with cyano groups and compounds containing COOEt groups, such as compounds 1versus3, 2versus4, 5versus6, 7versus8, 9versus10, 15versus16, 17versus18, and 19versus20, one can clearly see that all of these compounds but 9 and 10 obey the rule that the fungicide activity is improved when R1 was changed from a COOEt group to a cyano group. It was also found that the change of substituent on R2 could affect the fungicidal activity. Comparing the bioassay results of a number of pairs of compounds (1 and 4, 2 and 3, 5 and 7, 6 and 8, 10 and 11, 13 and 14, etc.) which have the same R1 group, but different R2, one can confirm that when R1 is a COOEt group, the introduction of PhCH2 into R2 plays a positive effect on the activities of the compounds. By contrast, when R1 is a CN group, compounds with CH3 moieties showed higher activities than those with PhCH2 groups.

In addition, the functional group diversity on R3 was also essential for the fungicidal activity of the title compounds. According to the data presented in Table 1 and Table 2, it can be observed that the introduction of a chlorine atom or fluorine atom on the substituted phenyl on R3 may improve the antifungal activity of the pyrazole derivatives. For instance, compound 12, (R3 with a 4-carboxylphenyl moiety) showed lower activity than compounds 3 (R3 is 2,3,5,6-tetrafluorophenyl), 6 (R3 is 2,4,6-trichlorophenyl), and 26 (R3 is 4-trifluoromethyphenyl). Heterocyclic moieties were also introduced at R3, but the N-containing and S-containing heterocycles do not seem to enhance the activities of the title compounds, despite the change of position of the heteroatom or the introduction of halogen atoms. All of compounds 1318, 21, 22 and 24 possessed low antifungal activities, for example.

Table 1. Fungicidal activities of compounds 132 at a concentration of 100 mg/L.

Comp.Inhibitory Rates (%)
B. cinereaR. solaniV. maliT. cucumerisF. oxysporumF. graminearum
177.86 e79.30 d72.09 d67.87 d70.63 e51.25 g
284.96 d98.02 b96.51 b88.78 c78.21 d91.00 c
396.50 b98.44 b98.28 b98.87 b93.05 b98.05 b
480.50 e93.38 c86.81 c85.73 c76.28 d73.00 d
557.00 h66.98 f58.14 f50.12 g53.21 h67.07 e
692.74 c97.25 b97.22 b96.13 b90.38 b98.05 b
778.24 e79.19 d88.89 c60.62 ef71.79 e71.25 d
885.00 d90.14 c94.19 b86.72 c71.79 e74.39 d
947.36 j71.97 e84.30 c51.51 g35.90 k30.49 k
1092.14 c96.45 b95.83 b94.77 b84.56 c93.17 c
1162.00 g37.91 k72.22 d58.11 f36.54 k26.83 m
1249.50 j66.98 f48.26 g47.04 h32.05 l35.37 j
1352.44 i64.10 g65.23 e39.27 i65.79 g47.04 h
1440.22 k60.93 h21.62 j13.37 k48.03 i33.78 j
1565.49 g58.47 h66.22 e64.68 e68.42 f

1. Introduction

Mango (Mangifera indica L.) is an evergreen fruit tree that is adapted to tropical and subtropical conditions. Mango cultivars vary considerably in fruit size, colour, shape, flavor, texture, and taste [1], and is cultivated in many regions of the world, including India, China, Pakistan, Mexico, Brazil, Egypt, and Nigeria [2]. In addition, mango production has increased in non-traditional mango producing areas including the UAE. According to the FAO (2014), UAE has significantly increased the cultivated area and the number of trees of mango (FAOSTAT; Available online http://faostat.fao.org/site/339/default.aspx), and growers have widely cultivated this crop due to its nutritional and economical values, and their delicacy in flavour and taste. Recently, mango has become an increasingly popular fruit in the UAE markets, after dates and citrus. Mango suffers from diseases worldwide caused by a variety of pathogens that affect all parts of the tree and, therefore, reduce yield and quality of the fruit [3,4,5].

Mango decline or dieback is a serious disease of mango. The causal agent of this disease remained uncertain for many years due to different fungi associated with it [4]. Fungal pathogens, such as Neofusicoccum ribis, Botryosphaeria dothidea, Diplodia sp., Pseudofusicoccum sp., and Ceratocystis sp. may infect mango trees individually, or in combinations, to cause mango dieback in different parts of the world [5,6,7,8,9,10]. Botryosphaeriaceae species, such as Lasiodiplodia hormozganensis, L. iraniensis, and L. egyptiacae have also been associated with mango dieback in Iran, Australia, and Egypt [10,11,12]. Lasiodiplodia theobromae (Syn: Botryodiplodia theobromae) [13,14], however, it has been reported as the causal agent in destroying mango orchards within days or a few weeks of infection in India, USA, Pakistan, Brazil, Oman, and Korea [15,16,17,18,19,20]. L. theobromae is a soil-borne wound pathogen that can affect all parts of the mango tree at all ages. Consequently, mango dieback is considered to be an important problem confronting the mango industry and marketing [21]. To date, the mango dieback disease nor its causal organism has been reported from the UAE.

The fungus, L. theobromae, often invades twigs and branches from their tips of mango trees causing them to dry and the plant to wilt [22]. Under favourable conditions, infections are characterized by dying back of twigs from the top, downwards, followed by discolouration and the death of leaves, particularly in older trees, which gives an appearance of fire scorch. Symptoms can also be observed on reproductive structures [23]. In severe situations, branches start drying one after another in a sequence resulting in death of the trees of the mango plantation. Commonly, once the symptoms of decline or widespread dieback are evident, it is difficult to stop or reverse the progress of disease. The disease has also been observed on different mango varieties associated with the variation in their susceptibility towards the fungus. Reports have shown that certain varieties are highly susceptible [24,25]. In vivo studies demonstrated that L. theobromae becomes aggressive in colonizing host tissues when plants are under abiotic stress, such as heat, water stress, or drought stresses [26,27]. In general, dieback is one of the deadly diseases of mango, which causes a serious damage to the tree and its productivity.

To manage dieback disease, traditional horticultural practices have been applied to confront the fungal attack. In general, avoidance of wounding of trees can limit disease incidence [28]. Infected parts should be pruned from 7–10 cm below the infection site, removed, and burnt [29]. Attempts to arrest early infections have been made by treating with copper oxychloride or pasting with cow dung on pruned ends [30]. Biological control (e.g., Trichoderma spp.) have also been tried to reduce disease incidence of L. theobromae under in vitro and in field conditions [31,32]. Implementation of integrated disease management (IDM) programs which combine cultural, chemical, and biological approaches are highly recommended to control mango dieback, reduce cost, and improve production efficiency. Despite its negative impact on the environment and human health, the use of chemicals continues to be the major strategy to lessen the menace of crop diseases. In this study, we report fungicide treatments against L. theobromae as an effective and reliable approach to reduce the economic losses associated with mango dieback disease. Growers in the UAE and other mango producing countries experiencing this damaging disease are expected to directly benefit from the outcome of this study. Future physiological and molecular analyses will shed more light on dieback disease and its causal agent, which will ultimately lead to the development of effective IDM strategies to manage this disease. Here, we aimed not only to determine the etiology of this disease on mango trees in the UAE, but also to evaluate some of the available fungicides for their effect on the pathogen under in vitro and in vivo conditions.

2. Results

2.1. Symptoms of Dieback Disease on Mango

Trees manifested with disease symptoms from Kuwaitat, Al Ain—in the eastern region of Abu Dhabi Emirate, UAE—were reported. The pathogen was observed to attack different parts of the mango trees. First, we noticed the disease symptoms in all plant tissues, including leaves, twigs, and apical tips. When the fungus attacks the leaves, their margins roll upwards (Figure S1) turning them a brownish colour (Figure 1A). Later, a scorch-like appearance developed, followed by the dropping of the infected leaves. Moreover, twigs died from the tips back inwards (toward the vascular tissues) (Figure 1A), giving a scorched appearance to the branches (Figure S1). We observed browning in the vascular tissues when longitudinal cross-sections were made in diseased mango twigs (Figure 1B). We also determined the disease symptoms associated with dieback on whole trees in the field.

At later stages of invasion, disease symptoms such as wilting, complete drying of leaves and death of the apical region of plants, may also appear (Figure 1C) and at different ages of mango trees (Figure S1). In general, branches dry out one after another in a sequence resulting in the eventual death of the whole tree. These symptoms on mango are typical of the dieback disease.

2.2. Morphological and Phylogenetic Identification of L. theobromae Associated with Dieback Disease

The isolate obtained on potato dextrose agar (PDA) and sporulation from naturally-affected tissues associated with dieback disease on mango trees (Figure 1A–C) were microscopically examined. On PDA, colonies of L. theobromae (Pat.) Griffon and Maubl. [13,14] had initial white aerial mycelia that turned greenish-gray mycelium with age (Figure S1). The mycelium produced dark brown to black conidia. We also observed mycelial growth and production of immature and mature conidia (Figure 1D). Immature conidia were subovoid or ellipsoid, thick-walled, hyaline and one-celled, turning dark brown, two-celled and with irregular longitudinal striations when at maturity. The size of mature conidia averaged 26.6 ± 0.51 μm long and 12.9 ± 0.28 μm wide. This suggests that L. theobromae is most likely the causal organism of dieback in mango.

We also established a phylogenic analysis of the isolate. PCR amplification of internally-transcribed spacers (ITS) of the rDNA gene from mycelium of infected tissues subcultured on PDA was carried (Figure 1). Our results detected the ITS gene of all infected tissues (Figure 2A), confirming that L. theobromae is frequently associated with all dieback disease symptoms on mango trees in the UAE. To check if the DNA sequences of this species collected in the UAE belongs to any isolated Lasiodiplodia isolate, we compared the identified strain with those available in GenBank based on a phylogeny tree. For that purpose, the ITS rDNA and the translational elongation factor 1-α (TEF1-α) gene [33] were used as a single gene set. The concatenated two-gene set (ITS and TEF1-α) were sequenced and deposited in GenBank (accession number: MF114110 and MF097964, respectively).

We also determined the relationship among this obtained and other closely related ITS/TEF1-α sequences [12,30]. All sequences were aligned and maximum likelihood analyses were performed for estimation of the phylogenetic tree. The adaptation to different plant hosts has led to the evolution of at least 13 cryptic species within the L. theobromae species complex [12]. The generated ITS/TEF1-α sequence belonging to our strain clustered in one clade corresponding to L. theobromae from different sources, confirming its identity with this species (Figure 2B). Among the studied Lasiodiplodia species, our analysis revealed that this pathogen is placed adjacent to L. theobromae CBS130989, distinguishing the obtained isolate from those belonging to other species of Lasiodiplodia, Diplodia, or Phyllosticta. Our phylogenetic analysis supports that the species L. theobromae (collection number DSM 105134) dominates in the UAE causing dieback disease on mango trees.

2.3. Pathogenicity Tests of L. theobromae on Mango Leaves, Fruits, and Seedlings

To confirm our results, detached leaves were spray-inoculated with the isolated pathogen. Following inoculation, a black rot developed on the leaves after five days post-inoculation (dpi) (Figure 3A). No disease symptoms appeared on control leaves sprayed with sterilized distilled water. Similarly, we inoculated mango fruits with the same pathogen. On fruits, dark brown to black lesions averaged 26.4 mm in diameter, beneath the PDA plugs containing the pathogen were observed at 5 dpi (Figure 3B). No disease symptoms were evident under the control plug without the pathogen. The symptoms of the disease were evident on the inoculated leaves (Figure 3A) and fruits (Figure 3B), but not from the control tissues, fulfilling the Koch’s postulates relating to the pathogenicity of L. theobromae (Figure 3C,D). Our data suggest that L. theobromae causes the disease on different tested tissues of mango.

In addition, we performed pathogenicity tests on healthy mango (cv Badami) seedlings, and monitored the disease progress. Plants were inoculated with 8 mm mycelial discs from 10-day old pure L. theobromae cultures grown in PDA, while control seedlings were inoculated with PDA without the pathogen. The seedlings were maintained under greenhouse conditions. Following inoculation, seedlings developed typical dieback symptoms showing a dark brown to black, necrotic tissues at the tip of the stem (point of inoculation). At the first week, black colour appeared on the stem at the site of inoculation (Figure S2). The disease progressed rapidly along the stem in the following weeks. At three weeks post-inoculation (wpi), symptoms often expressed as defoliated leaves and characterized by conidiomata development and tissue necrosis in inoculated plants (Figure 4A). At 5 wpi, seedlings showed complete black discolouration and necrosis of internal tissues of stems and branches (Figure 4B,C), forcing the leaves to fall (Figure 4D). Control leaf tissues remained symptomless. The pathogen was consistently re-isolated from the disease affected tissues; thus fulfilling Koch’s postulates that these detected symptoms were associated with the inoculation with the pathogen L. theobromae (Figure 4E).

2.4. In Vitro Evaluation of Systemic Fungicides Against L. theobromae

To evaluate the effect of fungicides, Score® (difenoconazole), Cidely® Top (difenoconazole and cyflufenamid), and Penthiopyrad® (Carboxamide), on the mycelial growth of L. theobromae, six concentrations, ranging between 25 and 1000 ppm of selected fungicides were applied in vitro (Figure S3). With the exception of Cidely® Top, there was significant difference among the concentrations of the other two tested fungicides below 250 ppm, in inhibiting the mycelial growth of the causal agent of dieback disease, L. theobromae (Figure S3). On the other hand, Cidely® Top fungicide increased fungal inhibition zone even at low concentration i.e., 25 ppm, and showed no, or slightly, significant deference when compared with other concentrations, ranging between 76–98% mycelial growth inhibition (Figure 5A). We also compared mycelial growth inhibition of L. theobromae in vitro at 250 ppm, which was considered as the most efficient concentration in the three fungicides. The results indicated that Score®, Cidely® Top, and Penthiopyrad® inhibited the mycelial growth and sporulation of L. theobromae by 77%, 92%, and 50%, respectively (Figure 5A,B). This suggests that the systemic fungicide, Cidely® Top, was the most effective fungicide at 250 ppm concentration among all tested fungicides; and that the fungal inhibition zones were observed, even when a low dosage of this fungicide was applied.