eChapters

X-ray diffraction (XRD) is an important experimental tool used in determining crustal structure of material system in bulk and nanoregimes. Therefore, it is important to understand the basic concepts of XRD.

Characterization Tools: Microscopy

Chemical Synthesis Methods

Several methods can be used for the production of these nanoparticles, namely: (i) top-down methods (Li et al., 2006) such as vacuum sputtering (Kuhn et al., 2002) or the decomposition of iron penta carbonyl (Fe(CO)5) in organic solvents (Karlsson et al., 2005); and (ii) bottom-up methods that promote the ‘growth’ of the nanostructures via chemical synthesis, for example through the reaction of iron(II) or iron(III) salts with sodium borohydride (Wang and Zhang, 1997). However, these methods present several limitations and problems; the top-down methods are generally expensive and require specific and costly equipment while the drawbacks of the bottom-up approaches are related to safety issues due to the toxicity of sodium borohydride, the production of flammable hydrogen gas during the process (Li et al., 2006), and the tendency to form large agglomerates, very fast and at a high extent, and therefore have a reduced reactivity and degradation efficiency.

Aim

To synthesize Zero valent (Fe) (nZVIs) iron using tea leaf extract.

Introduction

In this approach extracts of natural products (in most cases green tea leaves) with high antioxidant capacities are used. The compounds present in these extracts react with Fe (III) in solution to form nZVIs (Nadagouda et al., 2010).

Aim

Phytosynthesis of silver (Ag) nanoparticles using Cassia roxburghii aqueous leaf extract.

Introduction

The Ag nanoparticles constantly exhibit adverse effects on microbes such as inhibition and inactivation (Nasrollahi et al., 2011). The Ag nanoparticles synthesized by using various biological sources such as herbs, plants and biological organisms display excellent properties in various fields i.e., non-linear optics and intercalation materials for electrical batteries as optical receptors, catalysts in chemical reactions biolabelling and as antibacterials (Sanghi and Verma, 2009).

Aim

To synthesize gold (Au) nanoparticle using fungal mycelium.

Introduction

Biosynthesis of nanoparticles is a kind of bottom up approach where the main reaction occurring is reduction/oxidation. The fungal enzymes are usually responsible for reduction of metal compounds into their respective nanoparticles.

Aim

To synthesize phosphorus (P) nanoparticles from tricalcium phosphate using fungal mycelium.

Introduction

Green nanotechnology includes a range of processes that reduce or eliminate toxic substances to restore environment. Microorganisms like fungi and bacteria having naturally bestowed property of reducing and oxidizing nanoparticles, are used for synthesis.

Biological synthesis of Zinc Oxide (ZnO) nanoparticles

Aim

To synthesize nano ZnO particles using fungal isolates.

Introduction

The ZnO nanoparticle are effective anti-bacterial and antiodur agent. Biological synthesis of ZnO nanoparticle is a new approach for environmentally benign protocol in context to green nanotechnology, (Raliya and Tarafdar, 2013).

Polymer Clay Nanocomposites

Polymer clay nanocomposites has been used as to affect permeability of the maternal to gases and vapours. Majority of papers on nanocomposites have been focused on the use of smectitic type clays as nanoparticles. They are a group of swelling type of clay minerals including montmorilonite, nontronite, saponite, sauconite and hectorite.

A number of polymer-layered silicates (PLS) preparation methods has been reported in literature. The three most common methods to synthesize PLS nanocomposite are intercalation of suitable monomer and subsequent in situ polymerization, intercalation of polymer from solution and polymer melt intercalation.

In general, layered silicates have layer thickness on the order of 1 nm and a very high aspect ratio (e.g. 10–1000). A few weight percent of layered silicates that are properly dispersed throughout the polymer matrix thus create much higher surface area for polymer/filler interaction as compared to conventional composites. Depending on the strength of interfacial interactions between the polymer matrix and layered silicate (modified or not), three different types of PLS nanocomposites are thermodynamically achievable (Fig. 1 and 2 ) as outlined by Sinha Ray and Okamoto (2003). They are as follows:

Zerovalent (nZVI) Ionic Compounds

Although Ag⁺ contains a filled d¹⁰ shell, the structures of Ag compounds can in many instances be understood only if one assumes the existence of weak attractive Ag⁺ - Ag⁺ interactions. Similar attractive forces have been shown to be responsible for the formation of gold cluster compounds. In Ag⁺ solid state compounds the unoccupied 5s and 5p orbitals give rise to empty bands which may be partially occupied thus resulting in subvalent Ag compounds; examples are Ag₃O and Ag₂F. The compound Ag₅SiO₄ prepared from Ag and Si0₂ at 350 °C under a high pressure of oxygen, contains [Ag₆]⁴⁺ clusters (average oxidation state 2/3) and should be formulated at [Ag₆]⁴⁺ (Ag⁺)₄ (SiO₄ ^{4 -})₂.

Ag and Au forms numerous cluster compounds in which the metals are zero valent or subvalent. Examples are Ag₆ Fe₃ (CO)₁₂ {HC(PPh₂)₃} and paramagnetic cluster ions [Ag₁₃ Fe₈ (CO)₃ ²]^4-.

Introduction

Hydrogels are loosely cross-linked, highly hydrophilic, organic polymers. They can absorb and retain aqueous fluids up to 500 times of their own weight (Singh et al., 2011). In the field application, such absorbents exhibited a limited swelling capacity within the soil matrix.

Aim

To synthesize chitosan/nanosilica coating solution for fruit coating aiming at increasing shelf life.

Introduction

Application of edible coatings is promising to improve the quality and extend shelf life of fruits and vegetables (Lin et al., 2011). Chitosan, a versatile biopolymer derived from deacetylation of chitin had been widely applied in the fresh-keep field owing to its good biocompatibility, biodegradability, antibacterial activity and capacities to form film (Lin et al., 2011).

Aim

To synthesize silver (Ag) nanoparticle.

Introduction

The Ag nanoparticles easily interact with other particles and increases their antibacterial efficiency. The Ag nanoparticles can be synthesized using various methods: biological, chemical electrochemical, γ-radiation, photochemical, laser ablation etc (Udapudi et al., 2012). The most popular preparation of Ag colloids is chemical reduction of Ag salts by sodium borohydride or sodium citrate (Udapudi et al., 2012).

Aim

To synthesize iron (III) oxide (Fe₂O₃) nanoparticles

Introduction

Iron oxide nanoparticles have a number of applications in catalysis, sensors and high sensitive biomolecular magnetic resonance imaging (MRI) for medical diagnosis and therapeutics. They can be synthesize by the bottom-up approaches including the sol-gel technique.

Synthesis of Zn Oxide Nanoparticle

Aim

To synthesize Zn oxide nanoparticle

Introduction

Zinc oxide nanoparticle are effective anti-bacterial and antiodur agent. They can be synthesized by number of techniques including hydrothermal methods. Their synthesis using solvothermal process is described, (Aneesh et al., 2007).

Solid state materials can exists in various forms namely:

1. Single crystals (preferred for authentic characterization of structure and physical properties)
2. Polycrystalline powders that are agglomerates of large number of crystallites and are used for characterization when single crystal cannot be easily obtained
3. Nanocrystalline or nanostructured materials that posses large surface area
4. Thin films that can be grown in the best crystalline form for many applications
5. Amorphous systems that have no long-range translational order

History and Recent Developments

Richard P. Feynman in 1959 suggested that it should be possible to build machines small enough to manufacture objects with atomic precision. His talk “There is plenty of Room at the Bottom” is widely considered to be the foreshadowing of Nanotechnology. He delivered this talk at the annual meeting of the American Physical Society at California Institute of Technology (Caltech), In 1990, a team of IBM Physicists revealed that they could write (arrange) the letter IBM using 35 individual atoms of Xenon.

In 1992 a book entitled Nanosystems: Molecular Machinery, Manufacturing and Computation was published by Eric Drexler where he outlined a way to manufacture extremely high performance machines out of molecular carbon lattice (diamondoid).

Around year 2000, federal funding for Nanotechnology in the United States began with National Nanotechnology Initiative (NNI). NNI defined nanotechnology as dealing with materials with sizes between 1 and 100 nanometers exhibiting novel properties.

The Government of India, in May 2007 launched a mission on Nano sceince and nano technology (Nano Mission) with an allocation of `1,000 crores.

Leaf Spot

Colletotrichum capsici

Economic Importance

• The disease is common in many parts of South India, and in recent years has become very severe, causing much damage to the crop.
• The yield losses occur due to leaf spot infection is through the loss of photosynthetic green surface.
• During the period of heavy infestation, yield loss could be as much as 50%.

Damping Off

Pythium aphanidermatum

Economic Importance

• The disease is reported from China, Indonesia, South Africa and the USA.
• It causes extensive damage in the tobacco tracts of Andhra Pradesh, Maharashtra and Gujarat in India.
• The damage in some areas can be hundred percent, in the absence of protective sprays.

Wilt

Fusarium oxysporum f.sp. vasinfectum

Economic Importance

• Wilt is one of the major diseases of cotton, found wherever this crop is grown like Mexico, African countries, France, Italy, Yugoslavia, India and West Indies.
• In India, the disease was first reported from Nagpur by Evans in 1908.
• It appears in almost every cotton tract of the country. However, it is more or less absent in the loamy soils of Indo Gangetic plains and sandy loams of Maharashtra, Tamil Nadu, Karnataka and other States.
• In affected susceptible varieties yield losses exceed 45-50%.

Red Rot

Colletotrichum falcatum (Perfect stage: Physalospora tucumanensis)

Economic Importance

• The disease is so devastating in nature that it has been referred to as cancer of sugarcane.
• Red rot occurs in all sugarcane growing tracts of India and several epiphytotics have been recorded in the past from states like U.P., Bihar, Haryana, Punjab, Andhra Pradesh and Kerala.
• In U.P and Bihar, the two popular varieties, namely, CO 213 and CO 312 were badly hit by the epidemics of 1938-40 and 1946-47.
• There have been reports of red rot epidemics in many other countries viz., USA, Mauritius, Australia, Hawaii and Myanmar.
• Red rot also causes appreciable damage to canes in Bangladesh, Taiwan and Pakistan.
• The loss in cane yield has been estimated 12 to 44.5% and 35 to 86 % in cane yield of ratoon over preceding plant crops have different levels of disease.

Root Rot or Stem Rot or Charcoal Rot

Macrophomina phaseolina (Sclerotial stage: Rhizoctonia bataticola)

Economic Importance

• Root rot is an important disease of sesame as infection occurs from seed germination and emergence to adult plant stage.
• The losses incurred are even up to 100 per cent, if conditions are favorable.

Wilt

Fusarium oxysporum f.sp. carthami

Economic Importance

• The disease was reported for the first time from America in 1950.
• Wilt is a devastating disease infecting the crop right from seedling to maturity resulting in crop losses to varied degrees depending on the severity of infection.
• The disease incidence has been reported in India to the tune of 80 percent under favorable condition.

Wilt

Fusarium oxysporum f.sp. carthami

Economic Importance

• The disease was reported for the first time from America in 1950.
• Wilt is a devastating disease infecting the crop right from seedling to maturity resulting in crop losses to varied degrees depending on the severity of infection.
• The disease incidence has been reported in India to the tune of 80 percent under favorable condition.

Ozonium Wilt

Ozonium texanum var. parasiticum Thirum / Guizotia abyssinica

Economic Importance

• The disease was reported around Varanasi.
• Heavy losses are incurred as the diseased plants dry.

Wilt

Fusarium oxysporun lini

Economic Importance

• The wilt is very serious disease and occurs in almost all the countries where linseed is grown.
• Continuous cropping in the infected field makes the soil sick.
• Yield losses upto 80 percent has been reported due to this disease. However, in severe cases crop is destroyed completely.

Seedling Blight

Phytophthora parasitica

Economic Importance

• Seedling blight is wide spread in Africa, India and other Asian countries.
• The average economic loss due to the disease has been 10%.

White Rust

Albugo candida

Economic Importance

• White rust is an important disease affecting mustard crop in world.
• Countries where the disease occurs include the Brazil, Canada, Fiji, Germany, India, Japan, New Zealand, Pakistan, Turkey and UK.
• Up to 60 per cent or more yield losses on B. rapa and B. juncea from this disease have been reported in India.

Root Rot or Charcoal Rot

Rhizoctonia bataticola

(Pycnidial stage: Macrophomina phaseolina)

Economic Importance

• Charcoal rot is of great economic importance in the World and has been found to reduce head weight and diameter, stem height and weight and in particular, the stem girth and root weight of sunflower plants.
• The first report of the occurrence of charcoal rot on sunflower was from Sri Lanka by Small (1927).
• Under favourable condition, losses can reach 60-90%.
• Infected plants generally die before seed set is finished.

Tikka or Leaf Spot

Cercospora arachidicola/ Phaeoisariopsis personatum

Economic Importance

• Tikka disease of ground nut is regarded as the most important fungal disease of this crop.
• The infection appears when the plants are one to two month old; one can see this disease in the month of September in the plains of our country.
• Losses due to this disease may go up to 15-50%.

Dry Root Rot / Charcoal Rot

Macrophomina phaseolina

Economic Importance

• Dry root rot has been found in most of the soybean-growing areas of the India.
• The disease has been increasing in importance and can result in substantial yield losses.
• In most fields, stand losses range from less than 5% to over 50% in severe cases.
• It is most prevalent on seedling and young plants, causing a root and stem rot.

Anthracnose

Colletotrichum lindemuthianum, Glomerella lindemuthiana Shear [teleomorph]

Economic Importance

• Anthracnose is an important disease in Kenya, Uganda, Brazil, Argentina, Mexico, Guatemala, Costa Rica, Nicaragua, India, Peru, Ecuador, and Colombia .
• The disease causes huge losses in temperate and subtropical zones.
• Yield losses of 95% have been recorded in Colombia and over 92% in Malawi.
• Anthracnose also affects seed quality and marketability of the crop.

Anthracnose

Colletotrichum lindemuthianum (Sacc. & Magnus)

Glomerella lindemuthianum Shear

Economic Importance

• Anthracnose warrants a serious attention as it inflicts heavy economic losses to the crop, which may reach up to 100%, under conducive conditions.
• In India disease incidence has been reported to vary between 24.59 to 51.72 per cent.
• In Jammu region of Jammu and Kashmir state disease incidence of 63.46% was recorded.

Wilt

Fusarium oxysporum f. sp. lentis

Economic Importance

• The disease is present in countries such as India, Jordan, Nepal, Pakistan, Syria, Turkey, Russia, France, Hungary, Ukraine, Egypt, Ethiopia, Morocco, Sudan, Tunisia, Canada, Argentina, Chile and Uruguay etc.
• It may cause 5-10% yield losses but sometimes severe damage may result complete crop failure under favorable conditions.

Anthracnose

Colletotrichum lindemuthianum

(Sexual stage: Glomerella lindemuthianum)

Diagnostic Symptoms

• The pathogen can attack all aerial parts at any stage of plan growth.
• Main symptoms are circular, black, sunken spots with dark center and bright red orange margins on leaves and pods.
• In severe infections the affected parts wither off.
• Seedling get destroyed due to infection soon after seed germination.

Anthracnose

Colletotrichum lindemuthianum

(Sexual stage: Glomerella lindemuthianum)

Diagnostic Symptoms

• The pathogen attacks all aerial parts of plants and at any stage of their growth.
• Initial symptoms are circular, black, sunken spots with dark center and bright red orange margins on leaves and pods.
• In severe infections, the affected parts wither off.
• Seedlings get blighted due to infection soon after seed germination.
• The infected pods have discolored seeds.

Powdery Mildew

Erysiphe polygoni

Economic Importance

• Powdery mildew can be a serious disease of peas grown in India.
• It appears in epidemic form in the plains almost every year when the pods are formed in January-February.
• Since the disease occurs more frequently and covers a large host surface, it proves much more destructive than the downy mildew disease of the same crop.
• It is worst in dry weather as against the downy mildew, which flourishes in moist weather.
• Severe infections can reduce yield by 10–20%.
• Severe pod infection can lead to poor seed quality.

Wilt

Fusarium oxysporum f. sp. ciceris

Economic Importance

• Wilt is a serious disease of chickpea in India, Iran, Pakistan, Nepal, Myanmar, Spain, Tunisia, Morocco, Algeria, and Syria.
• Wilt is prevalent in most chickpea growing countries and is a major disease of economic importance in Asian countries.
• It was first reported in India.
• The yield losses encountered was reported to the tune of 60 per cent. However, it causes complete loss in grain yield if the disease occurs in the vegetative and reproductive stage of the crop.
• Seeds harvested from infected plants are lighter and duller than those from healthy plants.

Wilt

Fusarium udum

Economic Importance

• Wilt is widely prevalent throughout the world but more important in India and in eastern Africa.
• Wilt is the worst disease of red gram in India, causing severe damage wherever the crop is grown, especially in Maharashtra, Madhya Pradesh, Uttar Pradesh, Bihar and Tamil Nadu.
• The disease is reported to cause 30-100% loss in grain yield and may cause 100% yield losses in susceptible genotypes.
• The annual losses due to wilt have been estimated at $71 million in India.

Downy Mildew (Green Ear)

Sclerospora graminicola

Economic Importance

• Downy mildew is reported from temperate and tropical regions of the world.
• In India, the disease is a major stumbling block in the full exploitation of yield potential of hybrids.
• Several epidemics have been observed which resulted in substantial grain yield losses (upto 60 percent) in F₁ hybrids under South Indian conditions.
• Usually the losses in grain yield are directly proportional to disease incidence in the field.

Loose Smut/Kernel Smut

Sphacelotheca cruenta

Economic Importance

• Loose smut is distributed in Africa, Europe, Asia and America.
• In India, it occurs in the states of Andhra Pradesh, Maharashtra, Karnataka, Madhya Pradesh and Tamil Nadu.
• It is less common than grain smut.
• The pathogen affects plant growth and development adversely.
• Grain yield losses correlate well with percentage of the disease distribution in fields.

Southern Leaf Blight

Helminthosporium maydis (Syn: H. turcicum)

Economic Importance

• The first epidemics of the Southern corn leaf blight also known as Maydis leaf blight took place in the USA in 1970.
• A highly virulent strain called Race T appeared on maize hybrids with Texas male sterile cytoplasm.
• The disease development was related to the growing of maize with T-cytoplasm on large areas.
• Since 1990 the use of cultivars with T male sterile cytoplasm has been forbidden in selection.
• Presently, the cultivars with M and C types of sterility are used.

Rusts

The following three rusts are known to occur on barley

1. Stem black rust: Puccinia graminis tritici f. sp. tritici
2. Leaf rust: Puccinia striiformis
3. Leaf rust: Puccinia hordei

Of these, the first two are described under wheat and the disease symptoms are identical. These two rusts occur commonly on barley and cause severe damage in certain years.

Loose Smut

Ustilago nuda tritici (Ustilago tritici)

Economic Importance

• Loose smut has been observed and recorded on cultivated wheat from all over the wheat growing regions of the world.
• In India, it is prevalent in areas having cool, humid and semi humid climate during flowering period.
• Loose smut has recently became a more serious problem in the Ladakh region in the Himalayan mountains of India.
• If uncontrolled, loose smut can wipe out entire crops, since it replaces the grain.
• In areas where people depend on their grain crops for survival and don’t have the money or technology resources to control it, the disease can be devastating.
• Not only does it wipe out the crop, growers cannot even try again next year since any seeds they were able to harvest will be infected and will not produce seed the following season.

Fungal Diseases

Blast

Pyricularia oryzae (Sexual stage: Magnaporthe grisea)

Economic Importance

• Rice blast is the most important disease concerning the rice crop in the world.
• It has been found in over 85 countries across the world.
• The disease was first recorded in China in 1637.
• In India, occurrence of this disease was first reported from the Tanjore district of Tamil Nadu in 1918.
• The disease now occurs in almost all principal rice growing areas of India.
• Every year the amount of crops lost to rice blast could feed 60 million people.
• In severe cases losses amounting to 70-80% of grain yield was recorded.

A. Diseases

1. Bacterial Disease: Angular leaf Spot

Occurrence & severity status

Angular leaf spot disease, also known as ‘Black arm disease’ or ‘Bacterial blight’ is a disease of cotton potentially destructive in all cotton-growing areas of the world. The disease became prevalent in the USA during the 1950s (Schnathorst et al., 1960) and in India in the 1970s (Verma, 1986). In the USA, losses of between 34 and 59% were reported (Leyendecker, 1950; Bird, 1959). In India, losses of 5-20% were common in crops (Verma and Singh, 1971). Late and very late sowing of crops resulted in more yield reductions (Meshram and Raj, 1992). In general, losses due to bacterial blight in India are ranged between 1% and 27% depending on the cultivar and crop age (Mishra and Krishna, 2001). In Africa, data on crop losses is available for only a few countries. In Sudan, losses of 20% were common from severe infection (Last, 1960). However, nowadays, due to the wide availability of resistant varieties, its importance has been declined, with certain deviations where proliferation of new races has occurred as in Australia (Anonymous, 1980).

A. Diseases

1. Anthracnose

Occurrence & severity status

Anthracnose of sisal is also called as leaf spot or ring spot disease. The disease is frequently accompanied with sun scorch in Kenya and Tanganyika (Morstatt, 1930). This is considered as a leaf disease of minor importance, occurring on numerous species of Agave as well as the closely related genus Furcraea, including F. macrophylla Baker (wild sisal). The disease is distributed in warm temperate and tropical regions including southern Europe, British Guiana (Bancroft, 1914), the Neotropics, Cyprus (Georghiou and Papadopoulos, 1957), Kenya (Nattrass, 1961) and USA (Parris, 1959). It is also been reported to occur in various countries of Asia, including China (Tai, 1979), Korea (Cho and Shin, 2004), the Philippines (Teodoro, 1937) and Taiwan (Anonymous, 1979). In India, the disease was first reported as leaf spot caused by Colletotrichum agaves Cav. (Butler, 1905). Its occurrence was also reported from Antigua (Bancroft, 1910) and Puerto Rico (Macedo, 1943). Apart from sisal, the disease also occurs on tomato (Lycopersicon esculentum), potato (Solanum tuberosum) and over 35 other hosts, representing 13 families, chiefly in the Cucurbitaceae, Leguminosae and Solanaceae (Chesters and Hornby, 1965). The disease also occurs on onion and strawberry (Mordue, 1967).

A. Diseases

1. Anthracnose

Occurrence & severity status

Anthracnose has a serious impact on yield and fibre quality of flax (Linum usitatissimum) and is well-known in Europe, Asia and America. Flax anthracnose increased in Germany when flax production was expanding in the 1930s (Rost 1938). The anthracnose pathogen is seed- and soilborne, causes damping off of flax seedlings (Rost 1938), and is one of the causal organisms of so-called flax-sick soils (Bolley & Manns 1932). It has been recorded in nearly every country where the crop is grown either for fibre or oil. It is known to occur in Belgium, the British Isles, Canada, Formosa, France, Germany, Holland, Japan, Latvia, Lithuania, New Zealand, Poland, U.S.A. and the U.S.S.R. (including Siberia), in most of which countries its incidence has resulted in appreciable crop losses (Muskett and Colhoun, 1947). The disease is most common in cool and humid flax-growing areas (Nyvall, 1989). The disease is destructive to the crop, especially when the seed source is contaminated by the fungus. It can spread rapidly under favourable conditions causing severe local epidemics and heavy losses in yield and quality of both fiber and seed (Mercer, 1992c; Rashid, 2003b). The losses of 50% in fibre yield in an epidemic year were reported by Muskett and Colhoun (1947). Ondrej (1985) indicated a considerable loss in fibre strength on attack by anthracnose.

A. Diseases

1. Anthracnose

Hemp anthracnose is caused by two different fungi, viz., Colletotrichum coccodes (=C. atramentarium) and Colletotrichum dematium (Saccardo, 1882; Cavara, 1889).

i) Anthracnose (Colletotrichum coccodes)

Occurrence & severity status

The anthracnose disease occurs on many plants, including hemp (Cannabis sativa). The disease causing fungus is well-studied and it is an important pathogen responsible for black dot disease on potato. The fungus is an unspecialized pathogen, infecting a wide range of host families. It is most common in Canada and commonly regarded as a minor pathogen or secondary invader, but the fungus may seriously affect yield where intensive cropping without rotation occurs. It primarily affects the growth rate of plants and diminishes harvest. Moreover, it becomes fatal occasionally. Hoffman (1958, 1959) reported heavy hemp losses in central Europe. Conversely, Gitman (1968b) considered the pathogen was of little importance in the USSR.

A. Diseases

1. Alternaria leaf spot

Occurrence & severity status

In the years 2012 and 2013, black leaf spot disease was observed on ramie plants in Hunan and Hubei Provinces, China (Yu et al., 2016). Yu et al. (2016) claimed that their report was the first report of Alternaria leaf spot of ramie in China.

Symptoms

In the field, the symptoms of this disease included dark green to black big spots on leaves (Fig. 20A), often resulting in upwardly curled leaf margins (Yu et al., 2016).