3.1. Morphological Characterization of Fungal Isolates.
The citrus fruit samples were examined visually and screened based on typical symptoms of fungal diseases. A total of seven fruit-rotting fungal genera, including Alternaria, Botryosphaeria, Colletotrichum, Aspergillus, Fusarium, Penicillium, and Lasiodiplodia were isolated. These fungal species were identified based on their cultural and morphological characteristics and also subjected to molecular analysis. The identified fungal colonies were shown in Figure 3. Table 1 describes the diseases and isolated fungal pathogens, colony characteristics, spore characteristics, and the spore measurements.
Table 1: Morphological Characterization of Fungal pathogens isolated from diseased citrus fruits.
Disease
|
Isolated Fungal Pathogens
|
Colony Characteristics
|
Spore Characters
|
Spore size
|
Black mold rot
|
Aspergillus niger
|
The colony has a structure made up of tightly packed white or yellow conidial heads that are layered with a dense dark-brown coating.
|
Conidial heads are huge, globose, and dark brown in color. As they age, they become radiating and have a tendency to separate into multiple loose columns. Smooth-walled, hyaline, or becoming dark conidiophores are present. The phialides are carried on brown, frequently septate metulae and are biseriate with the conidial heads. Conidia are rough-walled, globose to subglobose, and dark brown to black in color.
|
Length = 19 – 30 µm
Width = 3.5 – 6 µm
Average = 24 x 5 µm
Conidiophore
Length = 200 – 223 µm
Width = 5 – 10 µm
Average = 208 x 7.6 µm
|
Alternaria brown rot
|
Alternaria citri
|
Colonies are quickly expanding, from suede-like to floccose, and black to olivaceous-black or greyish.
|
Simple, occasionally branched, short or elongated conidiophores generate multicelled conidia. Obclavate, occasionally ovoid or ellipsoidal, frequently with a short conical or cylindrical beak, smooth-walled, or verrucose conidia.
|
Length = 11 - 28 µm
Width = 8 - 15 µm
Average = 19.5 x 11.5 µm
Hyphae
Length = 33.5 – 52 µm
Width = 4 – 5 µm
Average = 43 x 4.5 µm
|
Green mold,
Blue mold
|
Penicillium italicum,
Penicillium digitatum
|
The colonies appear to be white, yellow, or pinkish, but they are typically green, blue-green, or grey-green. The majority of colonies have a silky to powdery texture.
|
A specialised conidiogenous cell known as a phialide produces the chains of single-celled conidia (macroconidia) in basipetal succession. Hyaline conidiophores can have smooth or rough walls. Spores are hyaline or greenish, smooth-walled or rough-walled, globose, ellipsoidal, cylindrical or fusiform, and green in color.
|
Length = 11 – 30 µm
Width = 7 - 26 µm
Average = 23 x 14 µm
Stipe
Length = 275 - 309 µm
Width =1.5 - 3 µm
Average = 268 x 2.8 µm
|
Fusarium rot
|
Fusarium oxysporum
|
Colonies are typically quickly expanding, light-colored or pale, and they may or may not have a cottony aerial mycelium. The thallus can be white, yellow, brownish, pinkish, reddish, or lilac in color.
|
Usually allow narrow phialides to produce both macro- and microconidia. Hyaline, two- to several-celled, fusiform or sickle-shaped macroconidia are the most common type, and they typically have an extended apical cell and pedicellate basal cell. Microconidia are hyaline, fusiform to ovoid, 1- to 2-celled, straight or curved, and pyriform.
|
Microconidia
Length = 3 - 7 µm
Width = 1.5 – 3.5 µm
Average = 5.8 x 2 µm
Macroconidia
Length = 15 - 22 µm
Width = 1.5 – 3.7 µm
Average = 18 x 2.5 µm
|
Canker
|
Botryosphaeria dothidea
|
Initially appearing olivaceous colonies that eventually turn dark grey and black. The mycelial mat has smooth borders and is relatively dense.
|
The ascospores are hyaline, unicellular, fusoid to ovoid in shape, and with tapering ends are the ascospores. Conidia are fusiform, narrowly or irregularly shaped, unicellular structures with rounded ends.
|
Length=17–22 µm
Width= 5.2-7.1 µm
|
Anthracnose
|
Colletotrichum gloeosporioides
|
On PDA, the colonies are cottony, initially white, and then turned greyish as the cultures grew older. They also have uniform borders in reverse and dark concentric zonation.
|
The spores are hyaline, one-celled, ovoid to oblong, slightly curved, or dumbbell-shaped conidia. Masses of conidia appear pink or salmon colored.
|
Length = 10-15 µm
Width = 5-7 µm
Average = 15 x 5 µm
|
Stem end rot
|
Lasiodiplodia theobromae
|
Initially white to light grey, the colonies eventually turned dark grey. The aerial mycelia appear fluffy from the upper side and black pigment appears on the reverse side of the plate.
|
While the adult conidia were thick-walled, dark brown, 2-celled (septate), and longitudinally striated, the young conidia were hyaline and aseptate.
|
Length = 22 - 37 µm
Width = 7 - 13 µm
Average = 30 x 9.5 µm
|
3.2. Pathogenicity of Isolated Fungal Pathogens. The results of the pathogenicity test revealed that the inoculated citrus fruit cultivars showed disease symptoms ranging from brown to black patches, sunken skin, and watery sores. On fruit samples, lesions were seen to form along the fruit stem, soften the fruit skin, and produce a watery pulp that can be pricked with a finger. Externally, symptoms seemed to be more severe (Figure 4).
After the inoculation for seven days, the disease rating scale for the three citrus fruit cultivars showed symptoms that were identical to those seen in fruits taken from orchards. Regardless of the isolate, disease symptoms appeared on all of the contaminated fruits. However, the severity of the diseases varied among the various fungus species the details of which can be seen in table 2.
Table 2. Disease rating scale on the citrus cultivars
Cultivar
|
Isolates
|
Percentages Rating scale
|
(0-1)
|
(2-5)
|
(6-10)
|
(11–49)
|
(50-100)
|
1
|
2
|
3
|
4
|
5
|
|
Control
|
+
|
₋
|
₋
|
₋
|
₋
|
|
CF-ANTH
|
₋
|
₋
|
₋
|
+
|
₋
|
|
CF-BMR
|
₋
|
+
|
₋
|
₋
|
₋
|
KINNOW
|
CF-BS
|
₋
|
₋
|
₋
|
+
|
₋
|
|
CF-SER
|
₋
|
₋
|
+
|
₋
|
₋
|
|
CF-GBM
|
₋
|
₋
|
₋
|
₋
|
+
|
|
CF-FR
|
₋
|
₋
|
₋
|
+
|
₋
|
|
Control
|
+
|
₋
|
₋
|
₋
|
₋
|
|
CF-ANTH
|
₋
|
+
|
₋
|
₋
|
₋
|
|
CF-BMR
|
₋
|
₋
|
₋
|
₋
|
+
|
|
CF-BS
|
₋
|
₋
|
+
|
₋
|
₋
|
SUCCRI
|
CF-SER
|
₋
|
₋
|
+
|
₋
|
₋
|
|
CF-GBM
|
₋
|
+
|
₋
|
₋
|
₋
|
|
CF-FR
|
₋
|
₋
|
₋
|
+
|
₋
|
|
Control
|
₋
|
₋
|
₋
|
₋
|
₋
|
|
CF-ANTH
|
+
|
₋
|
₋
|
₋
|
₋
|
|
CF-BMR
|
₋
|
₋
|
₋
|
+
|
₋
|
MORO BLOOD
|
CF-BS
|
₋
|
+
|
₋
|
₋
|
₋
|
|
CF-SER
|
₋
|
₋
|
₋
|
+
|
₋
|
|
CF-GBM
|
₋
|
₋
|
+
|
₋
|
₋
|
|
CF-FR
|
₋
|
₋
|
₋
|
₋
|
+
|
The data from the pathogenicity experiment was evaluated for equation 1 and subjected to one-way analysis of variance (ANOVA) by Minitab 2018 and Means were compared using Duncan’s test for means using SPSS. (Figure 5). According to the results of pathogenicity tests, inoculations resulted in irregularly shaped, roughly circular lesions on the surface of the fruits. All isolates produced lesions significantly (p<0.05) different from the control. The high rate of infections on Kinnow was caused by excessive growth of ANTH (anthracnose), BS (brown spot), and GBM (green blue mold), whose respective causal agents are Colletotrichum, Alternaria, and Penicillium sp. While the FR (Fusarium rot) has substantially enlarged lesion areas on Red blood, and the BMR (Black mold rot) induced by Aspergillus niger has a high lesion area on Succri. This showed that the Kinnow variety is more prone to fungal pathogens when compared with other varieties. Control fruit did not produce any noticeable outcomes
3.3. DNA Sequencing and Phylogenetic Analysis of Phyto-pathogenic Fungi. After DNA sequencing analysis, a total of 10 species belonging to the above-mentioned seven fungi genera were identified as the major fungal fruit diseases of citrus plants in the Khanpur orchards. The species were Colletotrichum gloeosporioides, Fusarium chlamydosporum, Fusarium oxysporum, Lasiodiplodia theobromae, Alternaria chlamydosporigena, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, Penicillium digitatum, and Botryosphaeria dothidea. These species along with their acquired accession numbers (Table 3).
Table 3. Fungal isolates of citrus retrieved from GenBank for the phylogenetic analysis
Isolate Code
|
Identity
|
Source
|
Host
|
Location
|
Accession number
|
Closest species
|
Similarity (%)
|
Isolate-CF-1
|
Colletotrichum gloeosporioides
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530857
|
Colletotrichum gloeosporioides KR911618.1
|
100
|
Isolate-CF-2
|
Fusarium oxysporum
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530858
|
Fusarium oxysporum KJ767070.1
|
100
|
Isolate-CF-3
|
Fusarium chlamydosporum
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530859
|
Fusarium chlamydosporum MN533778.1
|
100
|
Isolate-CF-4
|
Fusarium oxysporum
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530860
|
Fusarium oxysporum MG372014.1
|
100
|
Isolate-CF-5
|
Aspergillus fumigatus
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530861
|
Aspergillus fumigatus
MW332276.1
|
100
|
Isolate-CF-6
|
Lasiodiplodia theobromae
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530862
|
Lasiodiplodia theobromae KR092218.1
|
100
|
Isolate-CF-7
|
Alternaria chlamydosporigena
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530863
|
Alternaria chlamydosporigena
MN534774.1
|
100
|
Isolate-CF-8
|
Colletotrichum gloeosporioides
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530864
|
Colletotrichum gloeosporioides
MG543681.1
|
100
|
Isolate-CF-9
|
Botryosphaeria dothidea
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530865
|
Botryosphaeria dothidea
ON892080.1
|
100
|
Isolate-CF-10
|
Botryosphaeria dothidea
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530866
|
Botryosphaeria dothidea
MN634015.1
|
100
|
Isolate-CF-11
|
Aspergillus flavus
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530867
|
Aspergillus flavus
MG759550.1
|
99
|
Isolate-CF-12
|
Aspergillus niger
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530868
|
Aspergillus niger
LC133093.1
|
99
|
Isolate-CF-13
|
Aspergillus niger
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530869
|
Aspergillus niger
MG569605.1
|
99
|
Isolate-CF-14
|
Aspergillus flavus
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530870
|
Aspergillus flavus
MH329787.1
|
100
|
Isolate-CF-15
|
Aspergillus flavus
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530871
|
Aspergillus flavus
MH511139.1
|
100
|
Isolate-CF-16
|
Penicillium chrysogenum
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530872
|
Penicillium chrysogenum
MF475953.1
|
99
|
Isolate-CF-17
|
Penicillium digitatum
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530873
|
Penicillium digitatum
LC133090.1
|
100
|
Isolate-CF-18
|
Botryosphaeria dothidea
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530874
|
Botryosphaeria dothidea
KJ499737.1
|
100
|
Isolate-CF-19
|
Botryosphaeria dothidea
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530875
|
Botryosphaeria dothidea
MN634016.1
|
100
|
Isolate-CF-20
|
Lasiodiplodia theobromae
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530876
|
Lasiodiplodia theobromae
LC133088.1
|
100
|
Isolate-CF-21
|
Lasiodiplodia theobromae
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530877
|
Lasiodiplodia theobromae
MT302844.1
|
98
|
Isolate-CF-22
|
Aspergillus niger
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530878
|
Aspergillus niger
LC133093.1
|
99
|
Isolate-CF-23
|
Colletotrichum gloeosporioides
|
Fruit
|
C. sinensis
|
KPK, Pakistan
|
ON530879
|
Colletotrichum gloeosporioides
MT476872.1
|
100
|
Figure 6 displays the Neighbor–joining phylogenetic trees constructed using information from the ITS region of 5.8S-rDNA sequencing. Based on 5.8S ribosomal r-DNA sequencing the neighbor-joining phylogenetic trees were constructed for the isolates CF-1, CF-8 and CF-23 that were closely related to the Colletotrichum gloeosporioides strains KR911618.1, MG543681.1, and MT476872.1 respectively, whereas the fungal isolates CF-12, CF-13, CF-14, CF-15 and CF-22 were closely related to the Aspergilllus niger strains LC133093.1, MG569605.1, MH329787.1, MH511139.1, and LC133093.1 respectively. The fungal isolates CF-7 show close similarity with the fungal strain Alternaria chlamydosporigena MN534774.1. The isolated fungal pathogens CF-9, CF-10. CF-18, and CF-19 were closely related to the Botryosphaeria dothidea strains ON892080.1, MN634015.1, KJ499737.1, and MN634016.1 respectively. The fungal isolates CF-6, CF-20, and CF-21 were closely related to the Lasiodiplodia theobromae strains KR092218.1, LC133088.1, and MT302844.1. The fungal isolate CF-16 is closely related to the Penicillium chrysogenum strain MF475953.1and CF-17 is closely related to Penicillium digitatum strain LC133090.1. The isolated fungal pathogens CF-2 and CF-4 were closely related to Fusarium oxysporum strains KJ767070.1 and MG372014.1, whereas the fungal isolate CF-3 is closely related to the Fusarium chlamydosporum strain MN533778.1 respectively.
In all the phylogenetic trees, there were two primary groups. Above the branches are the bootstrap values. The sub-trees based on the evolutionary relationships of the isolates were produced by phylogenetic reconstructions of the ITS sequences. The Neighbor-Joining approach was used to infer the evolutionary history. The maximum likelihood method was used to estimate the evolutionary distances, which were expressed in terms of the number of base substitutions per site. Positions with holes and incomplete data were all removed. MEGA 11 software was used to conduct evolutionary analyses. To understand the evolutionary link between isolates of species, a thorough molecular phylogeny technique was used. All isolated species from citrus fruit showed genetic diversity according to phylogenetic research.
The fungal fruit diseases ought to be seen as complicated issues that call for systemic intervention strategies for control. The sensitivity of the detection method is less important than sufficient sampling techniques for the molecular detection of latent infections. Along with ecological and morphological information, DNA sequencing data can highlight differences within species complexes. As a result, it can be employed in the future to support species distinction. The findings of phylogenetic analysis and sequence alignment obtained by BLAST have shown that ITS sequencing is suitable for fungal isolates that are accurately identified up to the species level and further distinguished within the same species as specific strains, according to all phylogenetic trees with high bootstrap supports.
High genetic diversity in the fungal population provided clues about the infections' environmental compliance and fitness level. These variations are seen as a major threat to managing tactics like cultivar resistance and the administration of fungicides. According to the current work, ITS sequencing offers a quick, sensitive, and accurate way to identify the pathogenic fungus. In contrast to conventional methods, the ITS- sequencing method only requires a brief (3-5 days) incubation period for a fungal culture, making identification easier. The sequence analysis employing ITS sections is sensitive and sufficient for the identification of fungal diseases at the species level due to the high inter- and intraspecific variation and vast copies present in the fungal genome [29].
Within a week of incubation, fast growth rates were seen for all isolates at 25°C [30]. This is consistent with earlier research on the degradation of citrus fruits. Due to their capacity to produce resistant spores, Penicillium sp., Fusarium sp. And Aspergillus sp. are common. Aspergillus fumigatus was also referred to as a "dilemma for clinical management" because of its high level of resistance and environmental adaptability [31]. Fusarium is known to adore moisture and frequently develops on fruits with high moisture content, including several citrus varieties [32].
The quantity and quality of citrus fruits are deteriorating due to diseases caused by all of the recognized fungi. Nearly all isolates had an ITS fragment length of 580–600bp that was 95–100% similar, confirming earlier findings [33]. To assess a pathogenic Fusarium species that was affecting citrus plants in Greece, Italy, and Spain, ITS-rDNA. ITS markers are frequently employed to identify fungal infections and to ascertain their intra- and interspecific connections [29].
As the wooden crates are not properly stored in the local markets and the temperature is not appropriate, citrus fruit suffers from post-harvest stem end rot caused by Lasiodiplodia theobromae [34]. Lasiodiplodia theobromae has already been linked to citrus in reports from Pakistan [35]. In addition, four new species of Lasiodiplodia were found in citrus fruit in Iran, Pakistan's neighbor [36]. The variations in the isolates found by various researchers may be due to variations in the storage conditions of citrus fruits and the types of these products in the different areas where they are produced.
The different isolates discovered could be a result of the different geographic regions where the fruits are grown, which would then result in a different microflora there. Citrus fungal degradation is brought on by unfavorable storage conditions and their connection to nearby storage facilities [37]. The discovery of Aspergillus niger as a contributing factor in citrus deterioration in the present research is consistent with those of [38]. They proved their occurrence in fruits native to tropical humid climates and documented the predominance of Aspergillus species isolated from citrus fruit. Therefore, reducing fruit post-harvest losses is thought to be more environmentally friendly and sustainable than expanding the production areas to make up for these losses.
Numerous studies have used phylogenetic analysis to pinpoint multiple fungal species that are implicated in illnesses of citrus fruits [39-41]. For the pathogens that cause citrus fruit diseases to pose less of a threat to citrus production, human health, losses in fruit quality and quantity, and the nation's economy, it is essential to correctly identify the fungal isolates that are linked to these diseases.
The infection pathway of pathogenic fungus must be established because knowledge of the infection pathway may result in management strategies that lessen inoculum and host infection. Additionally, insects may contribute to the spread of some species [42]. Pruning wounds act as a primary source of entry for possible pathogens and increase tree stress [43]. Citrus trees are mechanically pruned in numerous commercial orchards across Khanpur and other places. Mechanical pruning frequently results in broken branches since the cuts are not always precise. Below the pruning wound, lateral branches emerge but frequently die. It seems that pathogenic Botryosphaeriaceae would have an ideal access route through the broken branches. The temperature has an impact on colonization's relative success as well. In the Lasiodiplodia theobromae complex, for instance, species are frequently discovered in warm tropical and subtropical environments [44, 45].
Previous cross-inoculation studies have shown that the temperature at which fruits are kept influences the dominance of one pathogen over another. In the present study, the fruit was kept at temperatures of 28-33°C which may have favored the development of lesions. In the present study, citrus fruit cultivars were used to prove and compare pathogenicity between strains that were isolated from citrus fruits with fungal diseases. All pathogens that were isolated from diseased sample citrus fruits produced lesions. The results revealed that the kinnow variety is more prone to fungal pathogens when compared with the other two varieties where green blue mold caused by Penicillium digitatum and Penicillium chrysogenum produced the largest lesions followed by Alternaria chlamydosporigena and Colletotrichum gloeosporioides. Similar results were reported by [46], who evaluated the pathogenicity of three different fungal strains, Penicillium digitatum, Aspergillus niger, and Rhizopus stolonifer, on Kinnow mandarin fruits and found that Penicillium digitatum was the most virulent strain, causing severe decay on the fruits. In a study conducted by [47], it was found that the fungal strains Colletotrichum acutatum and Colletotrichum gloeosporioides were pathogenic to citrus fruits. The researchers observed that these strains caused anthracnose symptoms in the fruit, which resulted in fruit rot and reduced fruit quality.
Overall, this study demonstrated that various fungal pathogens can be pathogenic to citrus, causing fruit and tree damage and posing an economic risk to the citrus industry by reducing fruit quality and overall yield. Thus providing valuable information on the pathogenicity of different strains of citrus.