INTRODUCTION
Preface
Citrus fruits, part of the Rutaceae family, are believed to have originated in Southeast Asia near Northeast India Notable species in this genus include lemon (C ×limon), lime (C ×aurantiifolia), sweet orange (C ×sinensis), sour orange (C ×aurantium), tangerine (C reticulata), grapefruit (C ×paradisi), citron (C medica), and shaddock (C maxima).
Citrus fruits are a vital source of vitamins, minerals, and dietary fiber, contributing significantly to nutritional health Rich in citric acid, these fruits are known for their sour taste, while some varieties are exceptionally sweet due to high fruit sugar content The primary commercial products of citrus trees include oranges and grapefruits, which are typically harvested when ripe, whereas lemons and limes are picked while still green Sweet oranges can be consumed fresh or processed into juice, with juice concentrates used for flavoring various beverages Notably, citrus juice is high in vitamin C, with a typical orange containing about 40 mg, compared to just 5 mg in an apple, making it essential for animal nutrition Since animals cannot synthesize vitamin C, a deficiency can lead to scurvy, a serious disease Historically, citric acid was extracted from citrus fruits for beverage flavoring, and it is now commonly used in soft drinks Additionally, the peel and juice of citrus fruits can be sweetened and made into spreads like marmalade, highlighting the economic importance of oranges beyond their fruit.
Citrus trees provide a variety of fragrant oils, primarily extracted from their flowers or peels, which are often byproducts of the orange-juice industry These aromatic oils are commonly used to scent many household products, including liquid detergents, shampoos, and soaps.
Citrus trees are among the primary crops in Vietnam, alongside traditional staples like rice and maize Renowned for their delicious flavor and rich nutritional profile, including high levels of folic acid, fiber, and vitamin C, citrus fruits have gained immense popularity both globally and within Vietnam.
Root rot disease manifests through the loss of feeder roots and results in above-ground symptoms such as reduced vigor and spindly growth Infested nursery soil exacerbates root rot, particularly in sensitive rootstocks, while orchards with susceptible rootstocks experience tree decay and yield losses As root death outpaces the formation of new fibrous roots, trees struggle to absorb sufficient water and nutrients, leading to diminished fruit size and production, leaf loss, and canopy twig dieback This disease poses a significant challenge to the expansion of citrus production, creating numerous issues for growers.
Root rot in citrus trees is primarily caused by various fungi, notably Armillaria mellea, Clitocybe tabescens, and Fusarium, along with oomycetes such as Pythium and Phytophthora Research indicates that Phytophthora spp and Pythium spp are significant contributors to root rot diseases (Timmer and Menge, 1988; A.H Thompson et al., 1995; BOZ Maseko and TA Coutinho, 2001) However, the role of Pythium cucurbitacearum in citrus root rot remains underexplored In Vietnam, limited studies on Pythium in citrus trees have resulted in insufficient information for effective management and control of this issue.
For the mentioned reasons, the research: "Morphological, biological,
The article "Molecular Characterization and Biological Control of Pythium cucurbitacearum Causing Root Rot Disease of Citrus Trees" highlights the importance of Pythium cucurbitacearum in relation to root disease affecting citrus trees in Vietnam It emphasizes the need for understanding the molecular aspects of this pathogen and explores potential biological control methods to mitigate its impact on citrus cultivation.
Objectives and requirements
This study aimed to identify and assess morphological, biological, molecular, and pathological characteristics of Pythium cucurbitacearum causing root rot disease of citrus trees to prevent and biological control
- To collect the infected soil samples from different locations;
- To isolate and identify from collected samples;
- To evaluate the morphological and biological, molecular characteristics of isolated;
- To evaluate the pathogenicity of pathogenic isolated;
- To evaluate the growth inhibition of antagonistic organisms to causative agents
LITERATURE REVIEW
Study in the world
2.1.1 The situation of citrus production in the world
Citrus fruits are among the most popular and widely cultivated crops globally, characterized by large shrubs or small to medium-sized trees with spiny branches and evergreen leaves Their flowers, often solitary or in small clusters, feature five (or sometimes four) fragrant white petals and numerous stamens, attracting pollinators through their scent and nectar The ability of Citrus species to hybridize easily aids plant breeders in developing new agricultural varieties by transferring desirable traits through controlled hybridization However, this hybridization complicates the work of taxonomists, leading to questions about the validity of some named Citrus species.
Citrus trees produce ripe fruit classified as a hesperidium, a type of berry characterized by a leathery peel known as the pericarp The outer layer, or exocarp, is referred to as the flavedo, while the middle layer, called the mesocarp, consists of the white, spongy albedo The innermost layer, the endocarp, contains locules filled with juice vesicles, or pulp, and string-like hairs that nourish the developing fruit Many citrus varieties have been developed to be seedless and easy to peel.
Global citrus production has more than doubled, rising from 58.9 million tons in 1981 to 121.3 million tons in 2014 Additionally, production increased from 123 million tons to 131 million tons between 2011 and 2016, as reported by the FAO.
In 2019, global citrus fruit production reached 144 million metric tons, with oranges representing approximately half of this total, making them the most widely traded citrus fruit The increase in citrus output in the early 21st century can be attributed to expanded cultivation areas, advancements in transportation and packaging, rising incomes, and a growing consumer preference for healthy foods For the 2019-20 period, world orange production was estimated at 76 million metric tons, with Brazil, China, India, the EU, the United States, Mexico, and Egypt being the leading producers.
2020) Although the COVID‐ 19 pandemic has affected on citrus production throughout the country, the citrus production in 2020 still increased
Figure 2.1 World production of citrus fruits in 2020, by region (in thousand metric tons) ( Source: M Shahbandeh, 2022)
Citrus fruits are cultivated in over 140 countries globally, with China, Brazil, India, Mexico, and the United States being the top producers Notably, China stands out as the leading producer of citrus fruits worldwide.
6 world China produced 44.6 million tonnes of citrus fruit in 2020, accounting for 28.21 percent of global citrus fruit production (FAO, 2021)
Oranges account for about 65% of global citrus production, with mandarins at 19%, lemons and limes at 11%, and grapefruit at 5% (Ismail et al., 2004) These fruits are not only tasty but also packed with nutrients, including sugar, citric acid, vitamin C, fiber, and pulp In the 2019/2020 marketing year, Brazil emerged as the top orange producer worldwide, generating 15.62 million metric tons, followed by China with 7.3 million metric tons.
Grapefruit and pomelo are citrus fruits believed to have originated in Southeast Asia The grapefruit, characterized by its yellowish or green skin, can weigh up to 0.5 kg China leads global production of these fruits, accounting for half of the total output, followed by the United States and Mexico.
2020) Lemon and lime are evergreen trees in the flowering plant The major lemons and limes-producing countries include Mexico, Argentina, and Brazil (FAO, 2020)
2.1.2 The stiuation of citrus root rot in the world
Root rot diseases pose a significant global threat to agricultural productivity, affecting various crops such as cereals, legumes, fruit trees, and tubers These pathogens lead to severe plant diseases, with symptoms often emerging below ground, making early detection difficult By the time above-ground symptoms appear, the yield has already suffered, putting plant health at risk Depending on the pathogen, host susceptibility, and environmental conditions, entire fields can be devastated Key symptoms include browning and weakening of root ends, root lesions of varying sizes and colors, root decay, and yellowing of the plant.
Root rot leads to symptoms such as leaf wilting, stunted plant growth, reduced yields, and potential crop loss (Liberato JR et al., 2014) This condition is exacerbated by various environmental factors, including excessive soil moisture, favorable temperatures for pathogens, soil compaction, inadequate drainage, continuous cropping, and other stressors affecting plants.
Root rot is primarily caused by pathogens such as oomycetes and fungi, with bacterial and viral infections being less common and less researched Additionally, root nematodes and other parasites can damage plants, creating entry points for further infections.
Oomycetes, or water molds, are a diverse group of eukaryotic organisms found in both terrestrial and aquatic environments, characterized by mycelial growth that resembles fungi However, molecular studies and distinct morphological features classify them within the kingdom Chromalveolata and phylum Heterokontophyta These organisms are notable for producing thick-walled sexual oospores, possessing cellulose in their cell walls, and exhibiting a vegetatively diploid state, along with heterokont flagella that include one tinsel and one whiplash type, as well as tubular mitochondrial cristae.
Pythium spp and Phytophthora spp are notable root pathogens among terrestrial oomycetes, exhibiting remarkable genetic plasticity that enables rapid adaptation to chemical control methods and host plant resistance.
Phytophthora spp and Pythium spp are the most prevalent pathogens causing root rot, with Phytophthora root rot being particularly severe in citrus plants Research by A H Thompson et al (1995) identified a strong association between Phytophthora nicotianae and Pythium spp in this context.
8 with diseased roots and rhizosphere soils caused feeder root rot with trees showing symptoms of citrus decline in the Transvaal Province of South Africa
All Phytophthora isolates were identified as pathogenic on citrus fruit and rootstocks, while Pythium spp were either avirulent or only mildly pathogenic The findings indicate that Pythium spp are not significant threats to citrus health and are unlikely to play a major role in the development of citrus root rot (BOZ Maseko* and TA Coutinho, 2002).
In recent years Phytopythium sp has gradually becom more popular, and studies have reported these causal agents on citrus trees Examples like
Phytopythium vexans causing root rot on Mandarin (Citrus reticulate L cv
Sainampueng) in Thailand of Noireung P et al (2020); Phytopythium helicoides causing stem rot of Shatangju mandarin seedlings in China ( Chen et al., 2016).
Study in the Vietnam
2.2.1 The situation of citrus production in Vietnam
Vietnam's tropical climate is ideal for cultivating citrus trees, including essential fruits like orange, pomelo, lime, and tangerine, each covering over 20,000 hectares These popular fruit trees thrive across all regions of the country, contributing to large-scale citrus production areas Notable regions include Ha Giang, Tuyen Quang, Hoa Binh, and Bac Giang for oranges and pomelos, while Bac Kan is known for its tangerines.
In the Bac Son district of Lang Son, the North Central region is renowned for its Nghe An oranges, particularly from Quy Hop, Thanh Chuong, Nghia Dan, Yen Thanh, and Con Cuong districts Additionally, Ha Tinh is known for its pomelo, especially from Huong Khe and Huong Son districts, as well as oranges from Vu Quang, Huong Son, Huong Khe, Can Loc, and Ky Anh districts In Thanh Hoa, notable orange varieties are found in Tho Xuan, Nhu Xuan, and Thach Thanh districts Moving to the Red River Delta, Hung Yen oranges thrive in Khoai Chau, Kim Dong districts, and Hung Yen city, while Hanoi is famous for its pomelo from Hoai Duc, Quoc Oai, Chuong My, and Phuc Tho districts.
In recent years, the area and output of citrus in Vietnam have shown significant growth From 2009 to 2019, the North experienced an average annual increase of 10% in citrus tree area, translating to approximately 7.3 thousand hectares per year Additionally, the output grew by 12.5%, reaching around 69.4 thousand tons annually By 2019, the total area dedicated to citrus trees in the North had reached impressive levels.
In Vietnam, citrus cultivation spans 122,000 hectares, representing 47.5% of the nation's citrus area and 29% of the total fruit tree area in the North The production of citrus fruits reached 1.98 million tonnes in 2020, a significant increase from 101,000 tonnes in 1971, reflecting an impressive average annual growth rate of 6.83% over the decades (MARD, 2020).
Table 2.1 The planted area and total citrus production in Viet Nam from
Year Planted area ( thousand ha) Productivity (thousand tons)
Source: Statistical yearbook of Viet Nam ( 2020), FAO ( 2020)
Hoa Binh province has 11,500 hectares of citrus trees, producing approximately 160,000 tons annually In Ha Giang, the orange crop year 2019-2020 saw an area of 9,117.7 hectares dedicated to oranges, with 7,133 hectares (78.24%) for local varieties and 1,984 hectares (21.76%) for Vinh oranges and others Over 6,200 hectares are ready for harvest, primarily in Bac Quang, Quang Binh, and Vi Xuyen districts, meeting high demand in Hanoi's market Hanoi itself has 14,244 hectares of fruit trees, including 2,400 hectares of pomelos and 579 hectares of Duong Canh oranges Citrus trees thrive along rivers like the Red, Lo, Gam, and Chay, and their cultivation is projected to be 4-5 times more economically efficient than rice farming.
Our country boasts a rich diversity of citrus species, particularly large citrus trees that have given rise to regions known for their unique local varieties Notable examples include the Xa Doan orange, Vinh orange, Canh orange, Tich Giang tangerine, Nam Roi pomelo, Dien pomelo, Thanh Tra pomelo, and Doan Hung pomelo.
In our country, orange and pomelo dominate citrus production, each covering approximately 38% of the total area, followed by lemon at 15.1% and mandarin at 8.6% In the Northern region, oranges represent nearly 45.6% of the citrus area, while pomelos account for 40.2%, tangerines for 7.4%, and lemons for 7.9% (MARD, 2020).
The heavy reliance on individuals and gardeners for the consumption of large quantities of products highlights the minimal role of businesses in this sector Additionally, the focus on selling crops fresh, with limited deep processing, often leads to complications during harvest In recent years, the market prices for oranges, tangerines, and pomelos have significantly declined, making them less attractive to consumers.
Oranges and pomelos are the most consumed citrus crops in the country, primarily grown in northern regions, with the domestic market driving demand for fresh produce In contrast, lemon cultivation spans approximately 9,600 hectares, representing less than 8% of total citrus land, yet it achieved an export value of $41.6 million in 2019, making up 94.4% of the citrus fruit export value.
The unchecked expansion of citrus cultivation, coupled with poor variety quality control and widespread pesticide misuse, has led to escalating citrus diseases This situation not only complicates disease management but also results in pollution and contamination of water and soil, ultimately compromising product quality.
The North is facing significant challenges in citrus production despite overall growth in area and output Predominantly local varieties, many of which are low quality, degenerate, and seed-heavy, hinder competitiveness in fresh markets and complicate processing Additionally, over 30 types of pests and diseases, particularly greening disease, yellow leaf, and root rot, threaten productivity and quality across all citrus-growing regions These issues not only impact orchard longevity and safety management but also escalate investment and maintenance costs Furthermore, the limited adoption of technical equipment and flaws in the farming process exacerbate these challenges.
The misuse of inorganic fertilizers and chemical pesticides poses significant risks to soil and water quality, leading to pest outbreaks and compromising food safety, which can erode consumer trust and drive prices down Growing oranges in hilly regions, particularly in the Northern Midland and Mountainous Area, presents challenges due to steep slopes and inadequate contour designs, complicating farming and harvesting efforts High production costs, stemming from low and unstable productivity, coupled with an inefficient value chain dominated by intermediaries, hinder farmers' competitiveness and profit margins Consequently, farmers, scientists, and government officials face numerous challenges in the development of citrus cultivation due to these systemic flaws and limitations.
2.2.2 The situation of citrus root rot in Vietnam
Root rot affects nearly all crops in Vietnam, such as pepper, coffee, durian, tobacco, and cassava Research by Truong, N.V et al (2008) identified Phytophthora capsici as the primary pathogen responsible for Phytophthora foot rot in black pepper, isolating it from diseased roots, collars, leaves, and root-zone soil.
Vietnam First report of Phytopythium vexans causing root rot disease on durian in Vietnam of L.D Thao et al (2020) This illustrates how common root rot is in agricultural production
According to the Plant Protection Department (2020), the area of orange trees affected by yellow leaf and root rot disease in the Northern provinces has reached 1,416 hectares, with 53 hectares experiencing severe impact, particularly in Ha Giang, Yen Bai, Hoa Binh, Bac Giang, and Nghe An Root rot hinders the plants' ability to absorb moisture and nutrients, leading to symptoms similar to drought stress and mineral deficiencies, including stunting, withering, and discolored leaves.
Root rot is a prevalent and serious disease affecting citrus plants, leading to withering foliage and shoots, ultimately causing the plant to die Contributing factors include excessive growth without proper planting, outdated cultural practices, improper production techniques, poor seed and seedling selection, and environmental conditions that favor the disease.
Pathogenic fungus of citrus root rot
Phytophthora species are a genus of oomycetes that resemble filamentous fungi in their morphology and habitat, yet they are phylogenetically linked to brown algae and diatoms within the kingdom Stramenopila While many heterokonts are unicellular flagellates, Phytophthora is multicellular and features a flagellated single-celled stage known as a zoospore in its life cycle The term heterokont refers to the unique shape of these cells, characterized by differentiated flagella—one whiplash and one tinsel—while the whiplash straminipilous flagellum is adorned with tripartite mastigonemes.
Oomycetes, similar to fungi, grow through fine filaments known as hyphae and can reproduce both sexually and asexually while absorbing nutrients from their environment Unlike fungi, oomycetes have cell walls made of cellulose and beta-glucans instead of chitin, lack cross-walls in their hyphae, and primarily exhibit a diploid life cycle Notably, Phytophthora species are known to cause diseases in plants, and nearly all oomycete species can produce disease symptoms in various plant hosts.
The genus Phytophthora is one of the most studied plant pathogens, primarily affecting herbaceous and woody dicotyledonous plants These soil-borne infections are responsible for a variety of diseases, including root, crown, and collar rots, as well as wilts, blights, and dieback in crops, ornamentals, and forest ecosystems globally Notable species such as P austrocedri, P capsici, P cinnamomi, P infestans, P kernoviae, P quercina, and P ramorum pose significant threats to agriculture and natural environments.
The Phytophthora genus has significantly influenced human and plant pathology, particularly during the 1840s when an "unknown agent" caused late blight in potatoes, leading to the devastating Irish Potato Famine of 1845 At that time, the origins of plant diseases like late blight were not understood, and the debate over the disease's source continues to this day.
The severity of the blight prompted scientists to investigate its causes, leading to the establishment of plant pathology as a scientific discipline In 1861, Heinrich Anton de Bary provided conclusive evidence that the "fungus" was the source of late blight The name Phytophthora is derived from the Greek words "phyton," meaning "plant," and "phthora," meaning "destruction," which translates to "plant destroyer."
Phytophthora species are identified through a combination of morphological features from both asexual and sexual phases, as well as colony morphology These species exhibit unbranched or branching sporangiophores, which can be categorized into umbellate, simple sympodial, and compound sympodial types Notably, Phytophthora litchii possesses a distinct form of compound sympodial sporangiophore that resembles the erect structure of downy mildew sporangiophores.
Figure 2.2 Life cycle of Phytophthora species (Source: G Abad et al., 2019)
In wet conditions, chlamydospores and oospores germinate to produce sporangia, which generate zoospores—single-celled swimming spores capable of moving through water on leaf surfaces or in waterlogged soil These zoospores are attracted to plant roots, where they form cysts upon contact Certain Phytophthora species can encyst and infect both leaves and roots The cysts then develop into hyphae, allowing the pathogen to invade plant tissues for nutrients Subsequently, Phytophthora reproduces by creating more chlamydospores asexually or oospores sexually, thus perpetuating its life cycle.
Pythium is classified as a member of the Pythiaceae family, order
Pythiales, class Oomycetes, phylum Oomycota, and kingdom Chromista in the
Pythium is a soil-borne pathogen comprising over 300 species, predominantly harmful to plants, and classified into ten distinct clades based on physical and genetic traits (Rossman et al 2017) This pathogen is prevalent in soil, sand, water sources, and decaying plant matter globally, affecting regions such as America, Asia, Africa, and Australia Pythium spp pose significant threats to various agricultural plants, particularly in orchards, causing common diseases like root rot and damping-off Notably, Pythium can infect plants during critical development stages, especially seeds before or during germination, and can attack new seedlings shortly after they sprout, leading to poor germination and root system damage.
Pythium species exhibit distinctive morphology characterized by mycelia that branch apically at right angles, with hyphae that are hyaline and typically 5-7 µm in width Newly formed hyphae often display visible protoplast streaming, and their walls are primarily composed of polysaccharides, lacking chitin or chitosan but containing 3-8% protein and 1-3% lipids (Postma et al., 2009) These fungi produce various forms of zoosporangia, including filamentous, lobulated, spherical, and oval types, although some species do not generate zoospores Swimming biflagellated zoospores develop within a thin, transparent vesicle, releasing numerous zoospores upon maturation in suitable aqueous conditions Oogonia exhibit diverse shapes, predominantly spherical and oval, with variations in surface texture, including smooth, rough, and spiny forms.
The life cycles of Pythium and Phytophthora root-infecting oomycete species share critical phases that are largely identical The accompanying figure illustrates the life cycle of a typical Pythium species.
Figure 2.3 Life cycle of a typical root Pythium species
(Source: Physiological and molecular plant pathogen book, 2003)
The formation of sporangia is a key feature of the asexual reproductive cycle in Phytophthora These sporangia can germinate directly in liquid or on surfaces to produce a germ tube, or they can undergo cytoplasmic cleavage to form multinucleate, biflagellate zoospores through indirect germination Typically, zoosporangia develop in aqueous cultures when temperatures decrease The released zoospores swim in search of host tissues such as seeds, roots, stems, or leaves, where they quickly encyst This process takes only a few minutes, after which the cyst germinates to form a germ tube that penetrates the host directly or via an appressorium-like structure The aseptate hyphae then spread throughout the plant tissue, creating a network of absorptive mycelium that leads to sporulation on the affected seedling, thus continuing the disease cycle by utilizing nutrients from the host.
The asexual phases of the pathogen can rapidly repeat throughout a plant's life, while the sexual cycle produces resilient oospores with thick walls that enable survival in harsh conditions Oosporogenesis involves the formation of a female oogonium and a male antheridium, which merges with the oogonium These dormant spores can overwinter in the soil and germinate in spring under favorable conditions, leading to the development of germ tubes that initiate the pathogen's asexual cycle once again.
The genus Pythium, established by Pringsheim in 1858, has been classified into 11 distinct clades based on DNA systematic analysis Significant morphological traits support the classification of these clades, with species in clade K demonstrating notable phylogenetic distinctiveness from other Pythium species.
Pythium spp exhibit characteristics similar to those of Phytophthora, and since the introduction of sequence-based phylogenetics, species in clade K have been distinctly classified Research by Villa et al (2006) demonstrated that Pythium species in this clade are closely related to Phytophthora, a finding supported by Bedard et al (2006) The Phytopythium genus represents a new group of organisms that, while structurally and biologically akin to Pythium and Phytophthora, can be identified by unique physical traits This genus comprises over 20 species, primarily saprophytic, but includes several that are particularly detrimental to plants, such as Pp litorale, Pp helicoides, and Pp vexans (Milosz Tkaczyk, 2020).
Most species have large, smooth oogonia, thick-walled oospores, and 1–2 elongate antheridia, laterally applied to the oogonium The phases in the life cycles of Phytopythium and Pythium are same
2.3.4 Comparison of Pythium species and Phytophthora species
Pythium and Phytophthora are two closely related genera within the Oomycota class, classified as fungal-like organisms in the Kingdom Chromista They share similar morphological characteristics, including coenocytic, hyaline, and freely branching mycelia, which are diploid and possess cellulose-based cell walls Both genera reproduce sexually by forming oospores and asexually by producing zoospores, which are heterokont with two laterally inserted flagella—one tinsel-like and the other smooth and whiplash Each oogonium contains a single oospore As significant soil-borne pathogens, they primarily affect bedding plants, leading to root rots and symptoms such as chlorosis, stunting, and wilting in infected plants.
Management Strategies for Root Rot
To effectively manage root rot, it is essential to plant resistant and tolerant cultivars The use of cultural practices, chemical treatments, and biological control agents plays a crucial role in this process Additionally, cultural activities can directly or indirectly influence soil-borne pathogen populations and the severity of root diseases.
Effective management of root rot infections involves several strategies, including draining wet soils, crop rotation, soil preparation through tillage, fertilization, and weed management prior to planting Planting at the optimal seed rate can reduce disease pressure by minimizing overcrowding, while crop rotation disrupts the disease cycle and alters soil chemistry Additionally, reducing the green bridge by eliminating weeds or volunteer plants is crucial, and herbicides like glyphosate can aid in this control Fungal pathogens can persist in the soil for years, complicating management and potentially requiring biocontrol agents Chemical treatments are typically not viable after visible above-ground damage occurs, making integrated pest management increasingly important This approach combines timely fungicide applications, crop rotation, soil moisture monitoring, and advancements in biocontrol, including the use of beneficial rhizobacteria like Bacillus pumilus and Pseudomonas putida, as well as antagonistic fungi.
The study investigates the impact of Chaetomium globosum, Chaetomium lucknowense, and Chaetomium cupreum, along with their crude extracts, on the regulation of the P nicotianae strain both in vitro and in vivo Application of spores and methanol extracts from various Chaetomium species to pomelo seedlings infected with P nicotianae in greenhouse conditions resulted in a significant reduction of root rot by 66% to 71%, while simultaneously increasing plant weight by 72% to 85%.
Conventional plant breeding and genetic engineering have successfully introduced disease resistance in various crop species A study on intergeneric somatic hybrid plants between Citrus and Poncirus trifoliata evaluated their resistance to root rot caused by Phytophthora parasitica The findings indicated that Page tangelo exhibited moderate vulnerability, trifoliate orange showed extreme resistance, while the somatic hybrids demonstrated resistance Root rot resistance is typically inherited across generations, and quantitative resistance involves multiple genes, highlighting the role of genetics in enhancing disease resistance.
MATERIAL AND METHOD
Objectives
Location and Time
Location: The citrus orchards was conducted in Hà Giang, Bac Giang and
Ha Noi The experiment was perfored at the laboratory and green house of the Plant Clinic – Vietnam National University of Agriculture
Material
- Rhisosphere soil samples, root samples
- Experiment instrument: microscope, petri disk, plastic, glass vaseá speculum, slides, electric scale, etc
- Cultural medium: WA, PDA, V8, SCDA, V8-, CDA and PA
- Liquid cultures: rain water, sterile rain water, 10% soil solution, sterile 10% soil soluion, sterile water
- Experiment chemical: Ethanol, distilld water, etc
- Antagonistic organism: Trichoderma asperellum; Bacillus velezensis, Bacillus sp CLNA, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12,
Methods
3.4.1 Collection of rhisosphere soil and root samples
The disease investigation follows the National Technical Regulation on the Surveillance Method of Citrus Pests, "QCVN 01-119: 2012/BNNPTNT," which was compiled by the National Technical Regulation Board on Plant Protection This regulation has been approved by the Ministry of Agriculture and Rural Development, as outlined in Circular No 63/2012/TT-BNNPTNT, dated December.
For the investigation of specialized citrus fruit cultivation, choose an area of at least 5 hectares to effectively represent the survey elements In contrast, for non-specialized cultivation, select an area of 2 hectares or more to adequately reflect the survey components.
Each survey element consists of 10 randomly selected points, positioned either randomly or along the diagonal of the investigation area It is essential that the survey site is situated at least one row away from the edge of the field, with one tree designated for each survey site.
4 holes for each tree in the canopy projection area, 30-50 cm from the edge of the canopy projection
Citrus tree samples exhibiting disease symptoms were collected from investigation sites for the purpose of isolating and identifying the causative agents It is crucial to select root samples that are either just beginning to show symptoms or are at an intermediate stage of development, as secondary rot and saprophytic microorganisms can complicate pathogen identification in later stages Additionally, soil samples should be taken from around the root samples Each sample bag must contain comprehensive information, including the collector's identity, specimen code, time of collection, degree of illness, and observed symptoms Furthermore, details such as the location, quantity of samples, species, orchard age, terrain, fertilizer type, and soil pH were meticulously documented.
To isolate Pythium, root and soil samples were collected from damaged orchards and stored in labeled plastic zip bags for laboratory transport The root samples were rinsed with tap water and surface-sterilized in 70% ethanol for 5-10 seconds before drying on filter paper Small root pieces, measuring 2-4 mm wide, were taken from the edges of lesions and placed on water agar (WA) The inoculated plates were incubated at room temperature and examined after 2-3 days Pure cultures were achieved by subculturing hyphal tips onto potato dextrose agar (PDA).
Soil trapping is an effective indirect method for isolating Pythium from soil samples, utilizing floating host leaves or fresh rose petals in a trapping solution This technique allows for the development of zoospores, which ascend to infect the host plant's leaves or petals if Pythium is present As a selective approach, soil trapping is particularly suited for isolating zoospore-producing species.
How to do it step by step:
1 Put about 100g of soil in a plastic cup
2 Pour sterile water or distilled water into the cup so that the soil is about
5 - 10 cm deep, stir well to dissolve the soil and let it settle
3 Drop fresh rose petals into the cup, these trap materials will float on the water
4 Put the cup in place for 2 - 5 days
5 Isolate the fungus after the disease has developed on the edge of the trap material, after rinsing in sterile water and decontaminating the surface, use
Root trapping method do the similar soil trapping
For pathogen identification, a sterile wire loop was utilized to transmit fungal tips onto PDA and V8 juice (20 percent Campbell's Vegetable) agar
3.4.3.1 CTAB Extraction of fungal DNA
1 Grow mycelia in pea broth culture 7-10 days or until sufficient mycelia
2 Harvest mycelia by vacuum filtration and freeze at -20° C
3 Add 150 àl Extraction Buffer, vortex Grind mycelia with sterile Knote pestle
4 Add 150 àl Nuclei Lysis Buffer and 60 àl 5% Sarkosyl, vortex to mix
6 Add 1 volume( 300 àl) Cloroform (CHCl3) : Isoamyl Alcohol (24:1),
7 Centrifuge 15 min, 12K rpm, room temperature
8 Transfer aqueous phase to a new microfuge tube Repeat chloroform extraction Centrifuge 15 min, 12K rpm, room temperature
9 Transfer aqueous phase to a new tube To aqueous phase add 0.1 volumes 3M Sodium Acetate (NaOAc), pH 8.0 and 2 volumes of cold 100% Ethanol
10 Allow DNA to precipitate in 30 min at -20°C
11 Cetrifuge to pellet DNA, 10 min, 12K rpm, room temperature Pour off supernatant
12 Wash pellet twice with 70% ethanol
13 Dry pellet in speed vacuum
14 Resuspend pellet in Te buffer, pH 8.0
Extraction Buffer 250 ml Per L Formula (FW)
Adjust pH to 7.5 with HCl Do not autoclave and store at 4°C
5% Sarkosyl: 5 g N-lauryl sarcosine per 100 ml H2O Autoclave
3M Sodium Acete 250 ml Per L Formula (FW)
Adjust pH to 8.0 with HCl and adjust volume to 1 liter Dispense and autoclave Store at room temperature
1 M Tris HCl ( pH 8.0) 100 ml per L Formula (FW)
Add Tris into 90ml H2O Adjust pH with HCl Bring to final 100ml volume Sterilize by autoclaving
0.5 M EDTA ( pH 8.0) 100 ml Per L Formula (FW)
0.5 M EDTA 18.6 g 186 C 10 H 14 N 2 O 8 Na 2 2H 2 O Add EDTA to 70 ml water and stir vigorously Adjust pH to 8.0 with NaOH Adjust to 100 ml volume and autoclave
10 mM Tris-HCl 0.5 ml of 1M Tris-HCl ( pH 8.0)
0.1 mM ETDA 0.01 ml of 0.5 M EDTA
10 mM Tris-HCl 0.5 ml of 1M Tris-HCl ( pH 8.0)
0.1 mM ETDA 0.1 ml of 0.5 M EDTA
3.4.3.2 Performing PCR/RT-PCR reactions
2 Ice (if there is no dedicated cooler)
Add to each PCR tube the following ingredients:
PCR Buffer (10X) 1.5 dNTPs (10 mM each) 0.3
3.4.3.3 Check the results of PCR/RT: PCR
Run agarose gel electrophoresis to check PCR/RT-PCR products
2 Make a 1% gel by weighing 1g of agarose into a vial and adding 100 mL of 1x TAE buffer to the vial.Microwave the vial to dissolve the agarose After the agarose has completely dissolved, add 1 L of Ethidium Bromide, shake well, and leave the vial at room temperature for about 60 °C
3 Apply pressure to the mold.Pour the gel into the mold (about 1 cm thick) Leave the mold at room temperature for 30 minutes to solidify the gel
5 Add 5 L of gel buffer to each tube (Loading Dye x6), mix thoroughly, and spin for 5 seconds
Run electrophoresis and check the results
1 After 30 minutes to let the mold at room temperature, proceed to withdraw the comb (hold the comb in the center, withdraw evenly so as not to break the well)
2 Place the entire gel mold tray in the electrophoresis bath, fill with 1x TAE buffer to cover the gel (gel depth is about 1mm)
3 Add 15 μL of PCR sample to the well In addition to PCR samples, always add 1 well of DNA marker (DNA marker) as standard
4 Plug in the power source for the electrophoresis machine, set the voltage to 100V and the duration of 30 minutes
5 After 30 minutes, turn off the electrophoresis machine, place the agarose plate on the UV generator and observe the results PCR product with primer pair BegoA For1/BegoARev1 will be ~1.2 kb in size
3.4.3.4 Extract DNA from GEL with the Total Fragment DNA Purification Kit
1 Cut the gel after electrophoresis with a razor 2 (Remove excess gel to limit the size of the gel)
2 Place up to 300 mg of gel in an eppendorf tube
3 Add 500 l of BNL Buffer/Plus to the sample and rapidly centrifuge
4 Incubate the tube at 55°C for 5–10 minutes, then shake for 2-3 minutes, until the gel is completely dissolved
5 Allow the mixture to cool to room temperature.Place the column in the collection tube
6 Fill the column with 800 l of the mixture.Centrifuge for 30 seconds at 11,000 x g, then discard the excess solution
7 Fill the Column with 750 l of Washing Buffer/Plus (with ethanol).Centrifuge for 30 seconds at 11,000 x g, then discard the excess solution
8 Centrifuge the column at full speed (18,000 x g) for 3 minutes to completely dry the membrane
9 Insert the new eppendorf tube into the column
10 Pour 40 l of elution buffer or distilled water into the column's membrane center and let it stand for 1 minute
11 To obtain DNA, centrifuge at full speed (18,000 x g) for 1 minute
3.4.4.1 Determine Pythium cucurbitacearum morphology causing root rot diseases on citrus trees
From all the isolates of fungal diseases, the three best purified samples from three provinces ( Ha Noi, Ha Giang, Bac Giang) were chosen to culture on
Mycelia expanded and eventually enveloped the dishes, with direct observations of the fungus on PDA media Examination under an optical microscope revealed distinct indicators of fungal growth on the PDA plates.
Besides from this method can be observed the characteristics of mycelium, sporangium formation and characteristics of sporangium
3.4.4.2 Evaluation of isolates growing in different culture media
Using 3 fungal sources, Pythium cucurbitacearum was cultured in the center of 90mm petri dishes ( 5 mm diameter holes) on 6 types of mediums and placed at 25°C Six types of mediums were tested:
- Potato dextrose agar ( PDA: 250 g of potato, 20 g of agar, 18g of dextrose per liter water)
- V8 juice with CaCO3 ( 200ml of V8 juice, 1000 ml water, 3 g CaCO3)
- V8- agar without CaCO3 (200ml of V8 juice, 1000 ml water)
- Carrot dextrose agar (CDA: 20 g of carrot root, 20 g of dextrose, 20 g of agar per liter of water)
- Sweet cassava dextrose agar ( SCDA: 200 g of cassava , 20 g of agar, 20g of dextrose per liter water)
- Pea agar ( PA: 150 g of pea seed, 20 g of agar, 5 g of dextrose per liter water)
Mycelium disks (5mm in diameter) of the pathogen were placed at the center of solidified culture media in petri dishes Daily monitoring of the pathogen's growth involved measuring the colony's diameter with a ruler until it fully covered the dish.
Each media were a treatment which had three replications
3.4.4.3 Sporulation in sterile water reservoir lakes of isolates growing in the different culture media
After seven days of growth on six different media, mycelium disks from the used pathogen were transferred to an empty plate Distilled deionized water was added to the agar level to promote sporangia production.
Daily monitoring of sporangia production included recording sporulation time and the quantity of sporangia formed The sporangia were rated on a scale from - to +++, where - indicates no sporangia production, + signifies low production, ++ represents medium production, and +++ denotes high production This assessment was conducted seven days after water was added.
3.4.4.4 Sporangia production of isolates growing in the different kinds of liquis cultures
Five kinds of liquis cultures were tested:
In there, the way to create 10% soil solution and sterile 10% soil solution are:
1 Pour 1 liter of water into 100 grams of soil and stir with a magnetic stirrer for 1 minute
2 Decant the upper portion through three layers of chessecloth to remove organic material after allowing it to settle for 5-10 minutes
3 Allow the suspension to settle for at least one night
4 Filter the upper part through one layer of filter paper, which would
34 need to be changed on a regular basis to guarantee efficient filltering The result is soil stock solution
5 Dispense soil stock solution into bottles and store in the refrigerator
6 To 100ml of full-strength soil stock solution, add 900ml of clean water Fill 250 mL bottles halfway with the solution and autoclave for 20 minutes Store at room temperature the 10% sterile soil solution obtainted
After seven days of growth on V8 juice medium, mycelium disks of the used pathogen were transferred to an empty plate, where various types of water were added to the agar to stimulate sporangia production Daily monitoring of sporangia production was conducted, with the quantity of sporangia formed rated on a scale from - to +++, where - indicates no production, + signifies low production, ++ represents medium production, and +++ denotes high production The ratings were assessed seven days after the water was added.
Calculation formula
Percentage of inhibition of radial growth (PIRG)
Using the following formula ( Siddiquee et al., 2009)
R1: growth radius in the control
R2: growth radius in the treatment
Statistical Analysis
Statistical analyses were performed using IRRISTART 5.0 and Excel
2010 All data were subjected to an analysis of variance (ANOVA) The mean values were compared using Fisher’s Least Significant Difference (LSD) test at p < 0.05
RESULTS AND DISCUSSION
Isolation of Pythium species
4.1.1 Results of fungual isolation from collected samples
A total of 42 soil and 20 root samples were obtained from Ha Giang, Ha Noi, and Bac Giang province affected orchards in 2021 and 2022 and used in the isolation of Pythium
Root portions from lesion edges were placed on Water agar and incubated at room temperature for 2–3 days Subculturing hyphal tips onto Potato dextrose agar and V8 juice resulted in pure colonies Soil samples were collected from severely affected citrus plants at a depth of 10–20 cm, utilizing a trapping method with fresh rose petals After three to five days, diseased tissue was aseptically taken from the junction of healthy and necrotic tissue on the inoculated rose petals and placed on WA media For pathogen identification, fungal tips were transferred to PDA and 20% Campbell's Vegetable agar Additionally, the experiment involved monitoring the rose petals and evaluating the infected ones under a microscope to observe their morphological characteristics.
Out of 62 samples collected, 21 were identified as having typical morphological characteristics of Pythium sp., indicating its role in the disease The remaining samples did not show any signs of Pythium sp The prevalence of citrus diseases is significant, with 33.87% of the total samples affected, presenting considerable challenges for production Detailed results of the sample collection and isolation can be found in Table 4.1.
Table 4.1 Results of collection and isolation of Pythium sp on citrus trees in and some provinces of Vietnam
No Symbol Location Source Morphologyl of pathogen agent No Symbol Location Source Morphologyl of pathogen agent
1 HG1 Ha Giang Soil - 32 HG22R Ha Giang Root -
2 HG2 Ha Giang Soil - 33 HG23 Ha Giang Soil -
3 HG3 Ha Giang Soil + 34 HG24 Ha Giang Soil -
4 HG4 Ha Giang Soil - 35 HG25 Ha Giang Soil -
5 HG4R Ha Giang Root - 36 HG25R Ha Giang Root -
6 HG5 Ha Giang Soil + 37 HG26 Ha Giang Soil -
7 HG6 Ha Giang Soil + 38 HG26R Ha Giang Root -
8 HG7 Ha Giang Soil + 39 HG27 Ha Giang Soil +
9 HG7R Ha Giang Root - 40 HG28 Ha Giang Soil +
10 HG8 Ha Giang Soil + 41 HG29 Ha Giang Soil +
11 HG8R Ha Giang Root - 42 HG30 Ha Giang Soil +
12 HG9 Ha Giang Soil + 43 HG30R Ha Giang Root -
13 HG9R Ha Giang Root - 44 HG31 Ha Giang Soil +
14 HG10 Ha Giang Soil + 45 HG31R Ha Giang Root -
15 HG11 Ha Giang Soil + 46 HG32 Ha Giang Soil -
16 HG11R Ha Giang Root - 47 HG32R Ha Giang Root -
17 HG12 Ha Giang Soil + 48 HG33 Ha Giang Soil -
18 HG13 Ha Giang Soil + 49 HG33R Ha Giang Root -
19 HG14 Ha Giang Soil + 50 HG34 Ha Giang Soil -
20 HG14R Ha Giang Root - 51 HG35 Ha Giang Soil -
21 HG15 Ha Giang Soil - 52 HG35R Ha Giang Root -
22 HG16 Ha Giang Soil - 53 HG36 Ha Giang Soil -
23 HG17 Ha Giang Soil - 54 HG36R Ha Giang Root -
24 HG17R Ha Giang Root - 55 LNM1 Bac Giang Soil +
25 HG18 Ha Giang Soil - 56 LNM2 Bac Giang Soil -
26 HG19 Ha Giang Soil - 57 LNM3 Bac Giang Soil +
27 HG20 Ha Giang Soil - 58 LNCC1 Bac Giang Soil +
28 HG20R Ha Giang Root - 59 LNCC2 Bac Giang Soil +
29 HG21 Ha Giang Soil - 60 UHD Ha Noi Soil +
30 HG21R Ha Giang Root - 61 UHR Ha Noi Root -
31 HG22 Ha Giang Soil - 62 UHV Ha Noi Root -
Note: - : None morphologyl of pathogen agent ; + : Exist morphologyl of pathogen agent
A Isolating from soil with fresh rose petals; B Brown lesion on rose petal after 2-3 days
4.1.2 Identification of Pythium species by Polymerase chain reaction (PCR)
Phytophthora, Pythium, and Phytopythium are soil-borne pathogens that cause root rot in citrus trees, often co-infecting plants within the same orchard Their similar morphological characteristics make differentiation challenging and unreliable However, the use of PCR for molecular analysis of nucleotide sequences from the internal transcribed spacer (ITS) regions of rDNA has proven effective in accurately identifying and distinguishing between Pythium species isolates.
I chose three samples to check whether the symptomatic samples collected from Ha Noi, Ha Giang, and Bac Giang were really infected by
Pythium PCR amplification was conducted with specific primers These fragments were cloned and sequenced The results are shown in Table 4 2 showed that 3/3 isolated samples were Pythium cucurbitacearum
Table 4.2 Results of PCR and ITS sequences of isolated LNCC2, HG11,
No GenBank fungi Host, country Accession
3 Pythium cucurbitacearum isolate R84 para rubber,
Figure 4.2 Electrolysis results of isolateted samples after PCR
4.2 Mophologycal and biological characteristics of isolates
To assess colony morphology, small plugs of active cultures grown for seven days on PDA were transferred For examining sporangia and zoospore release, Pythium cucurbitacearum cultured on the selective V8-Juice medium was moved to sterile water reservoirs after a full seven-day incubation Sporangia are located at the colony's edges, and by chilling the culture followed by returning it to room temperature, zoospore release can be induced This process should be repeated until sufficient zoospores are obtained.
Globose sporangia of Pythium cucurbitacearum are papillate, producing zoospores in a manner similar to Pythium A small discharge tube forms at the apex of the sporangium, allowing the release of its contents into a membrane vesicle at the tube's tip, where zoospores are differentiated and liberated The morphological characteristics of Pythium cucurbitacearum are detailed in the table below.
Table 4.3 Morphological characteristics of Pythium cucurbitacearum
Umbonate elevation; Filamentous form; Undulate margin; White color on PDA; rossette pattern on PDA
Dense cottony mycelium; Colorless, no branching, no septum
3 Sporangia Spherical shape with a distinct papilla
Aplerotic ( space between oospore wall and oogonium wall) or plerotic
Figure 4.3 Morphological characteristics of Pythium cucurbitacearum
A: Colony morphology of Pythium cucurbitacearum after 3 days on PDA media; B:
Mycelium morphology; C: Mature sporangia with a distinct papilla; D: Outgrowing papilla of sporangia; E: Oogonia with aplerotic oospore; F: Empty sporangia after zoospore released
Figure 4.3 Colony morphology of different isolateds on PDA media
A: Pythium sp LNM3; B: Pythium sp HG27; C: Pythium sp HG10; D: Pythium sp HG5 ; E:
Pythium sp HG9 ; F: Pythium sp HG30 ; G: Pythium sp HG31; H: Pythium cucurbitacearum UHD; I: Pythium sp HG29
4.2.2 The effect of medium on growth of P ythium sp
To optimize the sporulation and measure the mycelial growth of Pythium cucurbitacearum, six types of culture medium were tested: SCDA, PA, CDA,
V8, V8-, PDA with average temperature of 25°C The result as following the table below
Table 4.4 The diameter (mm) mycelium on growth of Pythium cucurbitacearum on different medium
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Chart 4.1 The diameter (mm) mycelium on growth of Pythium cucurbitacearum on different medium
PDA, or Potato Dextrose Agar, is a widely used microbiological growth medium derived from potato dextrose infusion, ideal for cultivating fungi and bacteria The nutrient-rich potato infusion supports mold sporulation, while dextrose enhances overall microbial growth.
Agar serves as a solidifying agent in this experiment, where after three days of incubation, the diameter of PDA reached 90 mm in treatments HG11 and UHD, while growth was slower in the LNCC2 source Pythium cucurbitacearum exhibited the fastest growth on SCDA and V8 media, achieving 90 mm in diameter within the same timeframe In contrast, V8 media showed the slowest growth, with diameters of 57 mm, 51.16 mm, and 58.83 mm, likely due to the absence of calcium carbonate, which may inhibit fungal growth due to the juice's acidity Additionally, PA and CDA are also preferred media for this organism.
Therefore, into six types of culture medium were tested, the optimize of mycelial growth for Pythium cucurbitacearum were SCDA, V8, and PDA media
Figure 4.4 Colony morphology of Pythium cucurbitacearum LNCC2
A: PDA media; B: SCDA media; C: V8 media; D: CDA media; E: V8- media; F: PA media
4.2.3 Sporangia production for Pythium cucurbitacearum of different medium
Pythium cucurbitacearum demonstrated robust growth across all six tested culture media To assess its sporangia formation capability, small plugs from active cultures, which had been growing for seven days on PDA, V8, V8-, SCDA, CDA, and PA, were transferred into sterile water reservoirs.
Table 4.5 Sporulation in water of agar disk of isolates growing in different culture media
Note: the level of sporangium quantity : - none; + low; ++ medium; +++ high
Research indicates that the development of sporangia from fungal sources varies significantly depending on the culture media used, with notable differences in both the quantity of sporangia produced and the time required for their appearance Among the tested media, the V8 medium demonstrated the highest efficiency in sporangia formation and the quickest formation time.
Sporangia formation was absent in CDA and SCDA media While V8 media did produce sporangia, the quantity was low and the time for spores to appear was prolonged In contrast, PDA medium exclusively yielded sporangia from the LNCC2 source All sources cultured on PA medium exhibited sporulation, producing more sporangia than both V8 and PDA, although the time for sporangia to appear was similar to that of V8.
The V8 medium is the optimal choice for the formation of the spore coat of Pythium cucurbitacearum, as it yields a higher number of sporangia compared to other media and facilitates the shortest formation time.
4.2.4 Sporulation in different kinds of liquid cultures
The results indicate that V8 medium is the optimal choice for the formation of the spore coat of Pythium cucurbitacearum In this experiment, V8 medium was utilized to promote sporangia production across various types of water, aiming to identify the most suitable liquid cultures.
Table 4.6 Sporulation in different kinds of liquid cultures of three sources on V8 medium
Note: the level of sporangium quantity : - none; + low; ++ medium; +++ high
The data indicates that the formation of sporangia in Pythium cucurbitacearum varies with different water sources Sporulation occurred in cultures using sterile 10% soil solution, 10% soil solution, sterile rainwater, and rainwater, with minimal differences in the timing of sporangium appearance across these liquid cultures Notably, sterile 10% soil solution emerged as the most effective medium, yielding the highest quantity of sporangia.
54 sporangia formation is absent As a result, it is ineffective for sporangium stimulation
The sterile 10% soil solution proved to be the most effective medium for the production of Pythium cucurbitacearum sporangia, as it yielded a higher number of sporangia compared to other liquid cultures.
Figure 4.5 Sporulation in different kinds of liquid cultures on V8 medium
A: The different kinds of liquid cultures ( the left to right : sterile water – rainwater- sterile rainwater- 10% soil solution- sterile 10% soil solution); B: The plate of experiment
Virulence tests – The lesions on Lime fruits, Orange fruits and Pomelo
Table 4.7 Diameter of the lesion (mm) after 3 to 5 days on Lime fruits, Orange fruits and Pomelo fruits caused by Pythium cucurbitacearum
Lime 5.00 a 5.00 a 5.00 a 5.00 d 5.66 c 6.66 f 5.00 d 6.00 d 6.00 f 5.00 c 5.33 d 5.33 e Tangerine 5.00 a 5.33 a 6.33 a 18.70 a 30.70 a 40.00 a 23.70 a 30.30 a 40.70 a 16.70 a 30.30 a 36.70 a Canh orange 5.00 a 5.66 a 6.00 a 14.30 c 18.00 b 24.70 c 13.30 c 17.70 c 26.00 c 12.30 b 16.00 c 20.00 c Vinh orange 5.00 a 5.00 a 5.00 a 15.00 bc 19.00 b 20.30 d 12.30 c 17.00 c 20.70 d 11.00 b 13.30 c 20.00 c Sanh orange 5.00 a 5.66 a 6.00 a 16.30 a 21.70 b 32.30 b 17.70 b 26.30 b 31.00 b 16.30 a 22.00 b 25.00 b Pomele 5.00 a 5.00 a 5.00 a 5.00 d 7.66 c 12.30 e 5.00 d 9.00 d 13.30 e 5.00 c 7.66 d 15.00 d
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Chart 4.2 The diameter of the lesion (mm) after 5 days on Lime fruits, Orange fruits and Pomelo fruits caused by Pythium cucurbitacearum
A statistically significant difference was observed in the diameter of lesions across all treatments Pythium cucurbitacearum caused brown necrotic lesions on all fruit types, with tangerines exhibiting the highest virulence and limes showing the least, characterized by smaller necrotic lesions In an experiment involving lime fruits, the diameters of necrotic lesions were measured as follows: control at 5.00 mm, Pythium cucurbitacearum LNCC2 at 6.66 mm, Pythium cucurbitacearum HG11 at 6.00 mm, and Pythium cucurbitacearum UHD at 5.33 mm The diameter of the lesions did not show a significant change compared to the original diameter.
For tangerine fruits, the diameter of necrotic lesions as follows: control, 6.33 mm; Pythium cucurbitacearum LNCC2, 40.00 mm; Pythium cucurbitacearum HG11, 40.70 mm; Pythium cucurbitacearum UHD 36.70 mm
The necrotic diameter increased rapidly from day 3 to day 5 The necrotic lesion
At day 5, the diameter of Pythium cucurbitacearum LNCC2 (40mm) was 2.14-fold greater than at day 3, attributed to the strong acidity of limes, which inhibits Pythium growth In contrast, tangerines and oranges, with a pH of 6, provide an ideal environment for fungal proliferation The thin peel of tangerines further exacerbates the impact of Pythium cucurbitacearum Notably, the necrotic lesion diameters for Canh oranges, Vinh oranges, and Sanh oranges were 26.00 mm, 20.70 mm, and 31.00 mm, respectively, likely due to their favorable pH levels of 6 and 5 In comparison, pomelo fruits exhibited the smallest lesion diameters caused by Pythium cucurbitacearum.
After 5 days, the diameter of necrotic lesions as follows: control, 5.00 mm;
Pythium cucurbitacearum LNCC2, 12.30 mm; Pythium cucurbitacearum HG11,
13.30 mm; Pythium cucurbitacearum UHD 15.00 mm
Infected wounded fruits exhibited brown necrotic lesions due to Pythium cucurbitacearum, highlighting the importance of minimizing damage to reduce yield loss and economic impact during and after harvest.
Figure 4.6 The brown necrotic lesions on citrus fruits causing by Pythium cucurbitacearum
A: lime fruits; B: Pomelo fruits; C: Canh orange fruits; D: Vinh orange fruits; E: Sanh orange fruits; F: Tangerine fruits
Pathogenicity test
Healthy plants are crucial for resisting pathogens from seedling to harvest, alongside favorable environmental conditions Minimizing damage to trees and fruits during harvesting is essential, as wounds can lead to increased vulnerability The pathogen Pythium cucurbitacearum thrives in optimal temperatures of 25–30°C and humid conditions, causing significant damage to citrus crops Isolates of this pathogen have been confirmed as the primary cause of such damage.
A pathogenicity test was conducted to evaluate the impact of Pythium cucurbitacearum on citrus plants The study involved artificial inoculation of wounded detached leaves, germinated seeds, and 2-month-old seedlings The experiments were designed in accordance with Kock's postulates to ensure accurate assessment of the pathogen's effects.
4.4.1 Evalution lesions caused by Pythium cucurbitacearum test isolates on citrus plants
A study isolating the cause of root rot from 62 disease samples identified Pythium spp in 21 samples, accounting for a frequency of 33.87% To definitively confirm Pythium as the infectious agent responsible for root rot, further experimentation is required, specifically infecting pomelo seedlings with the isolated Pythium.
During a two-month experiment, the environment was maintained at a low temperature of 8–15°C, which is below the optimal growth range of 25–30°C for Pythium cucurbitacearum As a result, the impact of the fungi on the plants remained minimal, and the plants developed normally, similar to those in the control treatment, without any visible lesions on stems or leaves Infected plants were only identified upon uprooting and assessing the roots, with dark brown tips or rotten roots indicating disease The results are summarized in the following table.
Table 4.8 The result of artifial inoculation of Pythium cucurbitacearum on pomelo seedlings
Treatment Symptoms of disease DI (%)
No symptoms, plants grow well; Roots develop normally with white root tips
The color of root tips change from white to brown, dark brown; Root rot appears on a few lateral roots first
The stems and leaves are completely normal
Root tips change color from white to brown to dark brown; root rot shows first on a few lateral roots
The stems and leaves are completely normal
The color of the root tips changes from white to brown to dark brown, and the first symptom of root rot appear on a few lateral roots
The stems and leaves are completely normal
All tested isolates of Pythium cucurbitacearum were found to be pathogenic to pomelo seedlings, causing brown to dark brown lesions at the root tips and resulting in root rot Additionally, there was no statistically significant difference in disease incidence among the different pathogen agent groups.
Figure 4.7 Evalution lesions caused by Pythium cucurbitacearum test isolates on pomelo seedlings
A: The experiment in greenhouse; B: The healthy seedling at treatment 1; C: The symptom root rot at treatment 2,3,4
4.4.2 Phathogenicity test on Pomelo detached leaves of Pythium cucurbitacearum
Table 4.9 The result of artificial inoculation of Pythium cucurbitacearum on
Diameter of the lesion (mm) after 7 days Color of lesion
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Treatment 1: Control ( PDA agar without Pythium condition onto the wounded leaf)
Treatment 2: PDA agar with Pythium cucurbitacearum LNCC2 onto the wounded leaf
Treatment 3: PDA agar with Pythium cucurbitacearum HG11onto the wounded leaf
Treatment 4: PDA agar with Pythium cucurbitacearum UHD onto the wounded leaf
The first symptom appeared after 3 days of inoculation of PDA agar with
Pythium cucurbitacearum was applied to wounded leaves, resulting in no change in lesion or color for the control treatment The pathogenicity of three isolates was demonstrated through detached leaves, which turned dark brown Treatments 2 and 3 involved PDA agar with Pythium cucurbitacearum LNCC2.
HG11 onto wounded leaf caused large lesions on leaves compared to treatment
4 There is no statistically significant difference between treatments 2 and 3 The result demonstrates that diseases caused by Pythium cucurbitacearum can show symptoms through leaves by changing color leaves and it can attack citrus thought wounded leaf
In a laboratory test, its potential to infect detached leaves was convincingly demonstrated (Fig 4.8 )
Figure 4.8 Phathogenicity test on Pomelo detached leaves of Pythium cucurbitacearum
A: The pathogenicity of three isolates was revealed by the detached leaves; B: The experiment conditions
4.4.3 Phathogenicity test on Pomelo germinating seeds of Pythium cucurbitacearum
Table 4.10 The result of artificial inoculation of Pythium cucurbitacearum on Pomelo seeds germination
The length of root rot After 7 days (mm)
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Treatment 1: Control (germinating seeds in V8 agar without Pythium condition) Treatment 2: Germinating seeds in V8 agar with Pythium cucurbitacearum
Treatment 3: Germinating seeds in V8 agar with Pythium cucurbitacearum
Treatment 4: Germinating seeds in V8 agar with Pythium cucurbitacearum
The study reveals that germinating seeds exposed to a pathogen-rich environment become infected, with treatments 2, 3, and 4 showing a 100% infection rate, and treatment 2 reaching an infection rate of 86.7% During germination, seeds absorb water and release exudates, making them vulnerable to infectious pathogens In the control treatment without pathogens, the germinating seeds exhibited white root tips, while infected seeds displayed dark brown root tips and signs of rot, indicative of root rot disease caused by Pythium.
64 cucurbitacearum The first symptom appeared after 2 to 3 days, and it demonstrated that the agent has high pathogenicity, and that germinating seeds have a low disease resistance and are rapidly infected
To ensure successful seed germination, it is essential to use disease-free seeds and maintain a clean environment during incubation, which helps prevent the attraction of pathogens, especially Pythium cucurbitacearum.
Figure 4.9 Phathogenicity test on Pomelo germinating seeds of Pythium cucurbitacearum
A: Germinating seed of control treatment; B,C: Germinating seed of Pathogen treatment
4.4.4 The influence of antagonistic organism on the causal agents
Plant pathogen biological control agents, or antagonists, are gaining attention for their role in developing alternative plant disease management systems Utilizing these microorganisms for plant protection presents a low-risk option for human health and, when combined with reduced fungicide use, helps protect the environment Soil-borne fungi and oomycetes pose significant threats to global crop production, particularly affecting citrus trees The effectiveness of biological control agents is largely due to their antagonistic properties and operational efficiency.
65 stimulating plant development and defensive systems
4.4.4.1 Growth inhibition of Bacillus velezensis, Bacillus sp CLNA, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with fungal pathogen
Bacterial and agent co-culture assay was performed to evaluate the antagonistic efficacy Pythium cucurbitacearum HG11 of bacteria (Bacillus velezensis, Bacillus velezensis, Bacillus sp CLNA, Bacillus sp TN1-KL1,
Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9) against Experiments were performed on PDA medium The results of the experiment are shown in the following table
Table 4.11 Growth inhibition of Bacillus velezensis, Bacillus sp CLNA, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with Pythium cucurbitacearum HG11
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Treatment 1: Control culture of Pythium cucurbitacearum HG11
Treatment 2: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Treatment 3: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Treatment 4: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Treatment 5: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Treatment 6: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Treatment 7: Simultaneous inoculation of Pythium cucurbitacearum HG11 and
Chart 4.3 The percentage of inhibition of radial growth ( antagonistic effect) of B velezensis, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp
YB12, Bacillus sp YB9 during in vitro with Pythium cucurbitacearum HG11
At treatment 1 ( control culture), Pythium cucurbitacearum HG11 on the 4th day was closely 90 mm in diameter, which shows the extremely fast growth
67 rate of mycelium In a culture experiment, B velezensis inhibited the growth of phytopathogenic mycelium as follows: day 1, 1.55%; day 2, 6.64%; day 3, 14.49%; and day 4, 16.41% Similar to other treatments, all antagonistic effects are low ( < 17%)
Observations of the mycelium of Pythium cucurbitacearum revealed significant growth in the presence of antagonist groups Within 3 days, the mycelium began to cover several antagonistic organisms, and by day 7, all antagonistic treatments were fully covered Consequently, the antagonistic effect diminished from 15.57% to 2.77% in treatment 3, indicating that all treatments with antagonists exhibited weak anti-mycelium activity against the phytopathogenic Pythium species studied.
Figure 4.10 Growth inhibition of Bacillus velezensis, Bacillus sp TN1-KL1,
Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with
A: Treatment 1; B: Treatment 2; C: Treatment 3; D: Treatment 7; E: Treatment 6 F:
4.4.4.2 Growth inhibition of Trichoderma asperellum on the Pythium cucurbitacearum during in vitro
Trichoderma species are highly effective biological control agents, utilizing both direct and indirect mechanisms to combat diseases Their antagonist activity arises from competition for nutrients and space, as well as the production of chemicals that inhibit spore germination, kill cells through antibiosis, or alter the rhizosphere's pH Mycoparasitism, a direct interaction with pathogens, involves physical contact and the secretion of hydrolytic enzymes, chemical toxins, and antibiotics that collectively eliminate the pathogen This study evaluated the effectiveness of Trichoderma asperellum on the growth of Pythium cucurbitacearum in PDA media, with results detailed in the accompanying table.
Table 4.12 Growth inhibition of Trichoderma asperellum on the Pythium cucurbitacearum during in vitro
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Chart 4.4 The percentage of inhibition of radial growth ( antagonistic effect) of Trichoderma asperellum on the Pythium cucurbitacearum
On the first day, the fungus Pythium cucurbitacearum exhibited normal growth levels, while Trichoderma asperellum showed no inhibitory effects However, by the second day, Trichoderma asperellum demonstrated significant inhibitory ability, with effectiveness recorded at 14.62%, 22.18%, and 22.6% in treatments 5, 6, and 7, respectively This resulted in a reduced mycelium growth rate compared to the control treatment.
Trichoderma asperellum on the 3rd day was 81 mm in diameter Antagonistic efficacy was recorded at the highest level in treatment 6 with 22.89%, treatment
The antagonistic efficacy of Trichoderma asperellum was found to be at a medium level across all three treatments (5, 6, and 7) Treatment 5 exhibited an efficacy of 21.44%, while treatment 7 showed a lower efficacy of 20.20% Notably, there was little change in the antagonistic efficacy of treatments 6 and 7 compared to the second day.
Figure 4.11 Growth inhibition of Trichoderma asperellum on the Pythium cucurbitacearum during in vitro
A: Trichoderma asperellum control; B: Pythium cucurbitacearum UHD - Trichoderma asperellum; C: Pythium cucurbitacearum HG11 - Trichoderma asperellum; D: Pythium cucurbitacearum LNCC2 - Trichoderma asperellum; E: Pythium cucurbitacearum UHD; F:
Pythium cucurbitacearum HG11; G: Pythium cucurbitacearum LNCC2;
4.4.4.3 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse condition
Among the tested antagonists, Trichoderma asperellum demonstrated the most reliable performance in in vitro conditions Consequently, I selected this strain to assess its effectiveness in managing diseases caused by Pythium cucurbitacearum in seedlings under practical conditions.
Table 4.13 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse
The length of rotten roots (mm) DI (%)
(at the same time: Pythium cucurbitacearum UHD-
(Pythium cucurbitacearum UHD – after 1 month add Trichoderma asperellum) 32.00 b 40.00
Note: Mean values in the same row with different letters are statistically different at the significant level α = 0.05
Chart 4.5 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse