Cross-Infection Analysis of Phytophthora spp. Isolated from Cacao (Theobroma cacao L.) Pods at Nagcarlan, Laguna, Philippines

Cross-Infection Analysis of Phytophthora spp. Isolated from Cacao (Theobroma cacao L.) 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Pods at Nagcarlan, Laguna, Philippines Ricci Julia De Mesa, Myles David Florano, Jarvic Louis Suguitan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6390079/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Despite its high demand, the production of cacao ( Theobroma cacao L.) remains low, which can be attributed to phytopathogens responsible for the destruction of cacao crops. Among these phytopathogens include Phytophthora spp. - pathogenic oomycetes that target plant species through their zoospores and cause black pod rotting. This research studied the ability of Phytophthora spp. to infect fruits commonly intercropped with cacao, such as papaya ( Carica papaya L.) and banana ( Musa spp.). Cacao pods exhibiting black pod rot symptoms were collected to isolate Phytophthora spp. from two cacao farms in Nagcarlan, Laguna, Philippines. Twenty-two isolates were identified as Phytophthora palmivora through their culture morphology, zoospore morphology, and molecular identity through ITS gene sequencing and confirmed through phylogenetic analysis. Five selected isolates were inoculated with their original host, cacao, and cross-infected with banana and papaya, which exhibited varying degrees of black pod rot symptoms. The lesions in cacao exhibited a higher degree of disease progression compared to banana and papaya. However, statistical analysis showed that bananas were more susceptible to the infection than papayas. Quantifying the degree of infection of P. palmivora from its host to commonly intercropped plants provides information on which plants are most vulnerable to infection within intercrop systems. Biological sciences/Plant sciences Biological sciences/Microbiology Biological sciences/Microbiology/Infectious disease diagnostics Biological sciences/Microbiology/Pathogens Black pod rot Cacao Intercropping Pathogenicity Phytopathology Philippines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Cacao is the raw material for the world-renowned delicacy known as chocolate. Despite a high demand for cacao, its production remains low [ 1 ]. Among many factors, phytopathogens contribute significantly to the low production of cacao. Phytophthora spp. are pathogenic oomycetes that target numerous plant species. They infect plant species through their zoospores, which are motile, asexual spores. Using their flagella, these can move according to physical, electrical, and chemical cues, allowing them to navigate to sites within the plant that they can infect, including the stomata, wound sites, and roots [ 2 ]. This mechanism of infection proved to be incredibly effective, with Phytophthora species having already caused numerous historical social crises during the 1800s, including the European Famine, the Great Famine of Ireland, and the Highland Famines. The species that caused these famines was identified as P. infestans , which originated in Central Mexico and was then unknowingly introduced to Europe, where it wrought devastation [ 3 ]. Despite causing such ravaging events in history nearly 200 years ago, the full extent of the genus Phytophthora is yet to be thoroughly studied, with research suggesting an estimated number of about 500 species of Phytophthora exist all around the world but are yet to be identified [ 4 ]. Having unknown species of Phytophthora out in the wild can have new implications, such as a far more extensive host range and varying degrees of pathogenicity compared to what is currently known [ 5 ]. This uncertainty, along with the dangerous precedent set two centuries ago for the ruin it can bring, is indeed enough to raise concerns, not only for cacao but also for entire farms that implement intercropping cacao with other potential hosts. Numerous studies have been conducted and published to identify unknown species of Phytophthora in the wild and understand their capacity to cause damage to different plant hosts. According to the United States Department of Agriculture’s research headed by Brasier in 2009, novel Phytophthora species are constantly being discovered worldwide, each adapting to different plant species as their hosts [ 4 ]. Phytophthora spp. are known for their diverse host range and highly virulent characteristics. Aside from cacao, other fruits are also devastated by various Phytophthora spp. Since cacao is usually intercropped with other plants, the chances of passing on Phytophthora spp. from one plant species to another is high. Upon an interview with the farmers from Nagcarlan, Laguna, Philippines, they revealed their usual intercrops with cacao, which include rambutan ( Nephelium lappaceum L. var. lappaceum ), banana ( Musa spp.), coconut ( Cocos nucifera L.), lanzones ( Lansium domesticum ), jackfruit ( Artocarpus heterophyllus ), avocado ( Persea americana ), and durian ( Durio zibethinus L.). This study focuses on bananas and papayas for convenience since they are of the optimum size and not seasonal. Tan (2021) wrote that cacao and bananas are promising intercropping duos because they use the same fertilizer and do not attract the same types of pests [ 6 ]. Previously, Phytophthora spp. was not viewed as a threat in bananas (Stover 1972) until Thompson (1981) recorded the presence of P . nicotinae B. de Haan var. parasitca (Dastur) Waterh. in a devastating Phytophthora finger rot in South Africa [ 7 , 8 ]. He described the symptoms as brown-margined with a firm black lesion on the peel and pulp, sometimes covered by a superficial, felty, white mycelium. Moreover, the possibility that bananas could be an alternate host for P . palmivora in cacao plantations was also reported [ 9 ]. In recent reports, P . cryptogea and P . inundata causing root and basal stem rot were detected on dwarf bananas in Italy [ 10 ]. Another good intercrop for cacao and banana is papaya [ 11 , 12 ]. However, papaya is a known susceptible host to Phytophthora . The known species that cause Phytophthora blight in papaya include P . palmivora and P . nicotinae . The symptoms of Phytophthora blight in papaya include water-soaked lesions with milky latex, fruit rot with white mycelium growth, stem cankers, and dark, rotten roots. Research conducted by Tocafundo et al. (2021) studied the direct and cross-infection of P. palmivora on cacao intercropped with papaya and peach palm [ 13 ]. It was found that while the derived P. palmivora isolates can infect other plant species, the specificity of the Phytophthora species meant that they were far more aggressive on their original host. While this means there would be less damage inflicted than anticipated in intercrop systems, it still exists. Moreover, given enough time to evolve according to their new host, the Phytophthora species may become just as pathogenic or even more. Local research was also conducted in South-Central Mindanao, where Phytophthora spp. isolated from infected cacao and durian trees were cross-infected with potential hosts, rubber and coconut. These four were used as these were often grown in intercrop systems in that part of Mindanao. The research found that P. palmivora was pathogenic to all the tested hosts [ 14 ]. Studies have made promising progress in combating Phytophthora infections in genetics. As zoospores infect hosts via taxis, researchers have avoided host recognition by silencing target genes, such as the Avr gene [ 15 ]. While this breakthrough was successful in the laboratory, it may take some time and more research before this method can be deployed in real-world applications. Furthermore, as the fear of cross-infection caused by Phytophthora within intercrop systems sets in, it may be misconstrued that the solution to the problem is monocropping, but it is not. As mentioned earlier, the Great Famine of Ireland was partially caused by the country’s dependence on potato and tomato monocropping [ 16 ]. With this situation in hand, this research investigated the possible cross-infection of Phytophthora species isolated from cacao collected within the local geographic region, Nagcarlan, Laguna, Philippines, to papaya and banana. This serves as a stepping stone in understanding these pathogenic species even more and learning which plants are in danger of infection within intercrop systems. Specifically, this study aims to isolate Phytophthora spp. from diseased cacao at Nagcarlan, Laguna, Philippines, and identify these through polyphasic methods. After successful isolation, the extent of cross-infection of these Phytophthora spp. was analyzed in banana and papaya. Results The collection conditions of the cacao pods were considered factors in successfully isolating the samples. During the cacao pod collection on October 22, 2023, a heavy downpour of rain ensued throughout the collection period at the collection sites in Nagcarlan, Laguna. From previous studies, it has been observed in other hot-humid countries in Southeast Asia that P. palmivora infects faster and spreads more during the wet seasons as the water brought on by the rain allows the flagellated zoospores of the P. palmivora to swim and infect nearby hosts [ 17 ]. In addition, the geographical structure of the two farms, while different from each other, was still a considerable factor in the success of isolating the samples. The steep slopes of the first farm help propagate the P. palmivora spores as water-carrying zoospores from infected plants at the top of the hill flow downward to the other plants at the bottom, promoting infection. The second farm has a flat and open field where water could accumulate on its surface, spreading disease around it [ 17 ]. Phytophthora palmivora Morphology Only five (5) isolates were described morphologically due to their high percentage identity. Table 1 shows the macroscopic and microscopic morphology of P. palmivora isolates in V8 agar. Overall, there were three major colony patterns observed: (1) stellate striated pattern (n = 3), (2) cottony with central rosette/chrysanthemum pattern and stellate radiate edge (n = 1), and a consistent (3) rosette/chrysanthemum pattern (n = 1). These patterns are congruent with the morphology of the isolated P. palmivora of Solpot and Cumagun (2022), whose isolation is done in the same country but in a different region [ 14 ]. The stellate pattern is typical in all Phytophthora species and not just P. palmivora as seen in the P. megakarya isolates of Akrofi et al. (2015) [ 18 ]. Moreover, the rosette or chrysanthemum growth pattern in P. palmivora was also reported by Solpot and Cumagun (2022) [ 14 ]. Still, this unique growth pattern can also be seen in the novel species of P. theobromicola sp. nov. reported by Decloquement et al. (2021) in Brazil [ 14 , 19 ]. Based on its colony growth, the CP1 colony can reach 35 ± 1 mm in 2 days, the CP2 colony can reach 36 ± 1 mm in 2 days, the CP3 and CP5 colonies can reach 40 ± 0.6 mm in 2 days, and the CP4 colony can reach 38 ± 0.6 mm in 2 days. Combined, P. palmivora isolates exhibited a growth rate of 38 ± 2 mm in 2 days. Table 1 Description of colony pattern, sporangia morphology, and chlamydospores morphology of the isolated P. palmivora. Isolate Code Identity Colony Pattern Sporangia Morphology Chlamydospores Morphology CP1 P. palmivora stellate striated pattern Shape: ovoid, globose, limoniform Length: 37 ± 5 mm Shape: globose Location: terminal Length: 38 ± 4 mm CP2 P. palmivora stellate striated pattern Shape: ovoid, globose Length: 24 ± 3 mm Shape: globose Location: terminal, intercalary, lateral Length: 29 ± 3 mm CP3 P. palmivora stellate striated pattern Shape: ovoid, globose Length: 28 ± 9 mm Shape: globose Location: terminal, intercalary Length: 30 ± 6 mm CP4 P. palmivora cottony with central rosette / chrysanthemum pattern and stellate radiate edge Shape: ovoid, globose Length: 30 ± 7 mm Shape: globose, semi globose Location: terminal Length: 28 ± 5 mm CP5 P. palmivora rosette / chrysanthemum pattern Shape: ovoid, globose, ellipsoid, limoniform Length: 28 ± 7 mm Shape: globose Location: terminal, intercalary Length: 30 ± 4 mm There was also variation in the sporangia shape, but all isolates uniformly presented common shapes, which are ovoid and globose with evident papillae. Kuswinati et al. (2023) reported a varying papilla of Phytophthora sporangia, which can be papilated or non-papilated [ 20 ]. However, the ovoid sporangia shape was the most abundant of all isolates. This is also true for the P. palmivora isolated by Alsultan et al. (2022) in Malaysia [ 21 ]. Moreover, CP1 and CP5 showed a limoniform sporangia shape, and CP5 showed a unique ellipsoid sporangia shape. The lengths of all sporangia vary even within the same isolate. In terms of chlamydospores, all isolates showed uniform terminal globose chlamydospores, while some isolates showed intercalary and lateral chlamydospores. Some chlamydospores showed a semi-globose shape. All these morphological characteristics of chlamydospores are common in P. palmivora and other Phytophthora spp., as reported by Kurbeti et al. (2020), Scanu et al. (2021), Alsultan et al. (2022), and Kuswinati et al. (2023) [ 22 , 23 , 21 , 20 ]. Different Phytophthora spp. have similar morphological characteristics, while the same species of Phytophthora may show varying morphological characteristics. Thus, morphological characterization is only helpful in identifying Phytophthora at the genus level. Several studies also report variations in colony morphology and microscopic morphology between the same species of P. palmivora [ 24 , 22 , 21 , 20 ]. Molecular and Phylogenetic Analysis Following the isolation of the samples from the cacao pods, molecular identification of the samples was started simultaneously with the morphological analysis. The samples were sent to the Plant Pathology Department of the University of Florida for DNA sequencing. Out of the twenty-two isolates sent, twenty-one were revived successfully following the delivery of the samples overseas, which were then sequenced. The resulting sequences sent back were cross-checked with sequences from the NCBI GenBank for identification. It was found that all twenty-one isolates were identified as Phytophthora palmivora . From those, five isolates that presented with high percent identities were chosen as representatives for this study, which are shown in Table 2 . The selected five isolates, CP1, CP2, CP3, CP4, and CP5, all matched with P. palmivora isolate UHO-1 (MT644188.1) on NCBI’s GenBank [ 25 ] with 99.39%, 99.39%, 100%, 99.85%, and 99.23% percent identities, respectively. Table 2 Molecular identification of the five isolates. Isolate Code Identification Percent Identity Accession Number CP1 Phytophthora palmivora 99.39% MT644188.1 CP2 99.39% CP3 100% CP4 99.85% CP5 99.23% These sequences then underwent phylogenetic analysis with the use of MEGA X [ 26 ], along with additional reference sequences for comparison retrieved from NCBI’s GenBank, including ten reference sequences of P. palmivora isolated from Theobroma cacao (isolates 227, TARS1, I320, CNC-P8, UHO-1, PPM1, H12, Co-17, CPR22, and CBS179.26), two reference sequences of P. palmivora isolated from Carica papay a (isolates CHA134 and BRIP 40506a), five reference sequences of Phytophthora sp. from Type Material: P. litoralis, P. multivora, P. plurivora, P. capsici , and P. tropicalis , two reference sequences of Phytophthora sp. isolated from Musa sp . ( P. cryptogea PHBan1 and P. inundata PHBan3), and one reference sequence of Pythium zingiberum (isolate UOP389) as the outgroup. The resulting Phylogenetic Tree is shown in Fig. 4 . All the formed clades in the Phylogenetic Tree are strongly supported, with bootstrap values ranging between 98–100%. Starting from the bottom of the tree, the Phytophthora sp. clade can be seen diverging from the Pythium zingiberum by a large margin, which is to be expected from the outgroup. Among the referenced Type Material Phytophthora sp . isolates, P. tropicalis and P. capsici were the first to diverge away from the group, which was then followed by the clade containing P. plurivora and P. multivora . The remaining isolates were then divided into two groups; the first comprised of P. cryptogea and a minor clade containing the P. inundata and P. litoralis . The second clade comprised the P. palmivora isolates, including the PUP Cacao Isolates. Pathogenicity Test and Cross-Infection Test The results of the pathogenicity and cross-infection tests of the five isolates of Phytophthora palmivora from infected cacao are shown in Table 3 . Two isolates infected unripe cacao pods with varying degrees of severity. Brown-reddish lesions were first observed on the third day of C1T2 with an average diameter of 50mm. On the fifth day, all replicates of C1 varied in the severity of infection, showing brown-reddish to dark brown lesions with the appearance of mycelia and spores over them. Concurrently, dark brown lesions were observed around the wound of C3T2. The average lesion diameter for infected cacao on the fifth day ranged from 29 mm to 61.78 mm. On the seventh day, all replicates of C1 showed widened and darkened brown lesions with increased manifestation of mycelia and spores. At the same time, two replicates of C3 showed varying degrees of infection; C3T1 exhibited an irregular brown lesion near the wound, while C3T2 presented a spread-out dark brown lesion with an increased amount of mycelia and spores. The average lesion diameter for infected cacao on the seventh day ranged from 55 mm to 99 mm. The lesion description of the cacao pods for the third, fifth, and seventh-day are elucidated in Supplementary Table 1. None of the negative control setups developed lesions. Table 3 Mean lesion diameter of Phytophthora palmivora isolates on cacao, banana, and papaya after 7 days of inoculation. *Means within the same column followed by a common letter are not significantly different at p < 0.05 using Tukey’s Studentized Range (HSD) test. The mean of lesions was calculated with three independent replicates per sample unit. Isolate Code Inoculum source Cacao (7th day) Banana (7th day) Papaya (7th day) CP1 Phytophthora palmivora (cacao) 99 a 13 a 9.11 ab CP2 9 b 9.89 b 8 ab CP3 55 ab 10.78 ab 8.67 ab CP4 10.33 b 10.67 ab 8.5 ab CP5 9.22 b 11.78 ab 11.5 a CONTROL - 5 b 5 c 5 b Following the measurement of the lesions on Day 7, the diseased cacao pods were sliced open horizontally to visualize the cross-section of the cacao pods after being infected by P. palmivora . C1T2 was opened to reveal some mild shrinkage of the cacao’s pulp and browning of the placenta. With this cross-section, the spread of the P. palmivora -induced lesion on the 7th day is also visualized, and it was observed to have encompassed the entire cacao pod’s pericarp. C1T3 was also opened to reveal an entirely devastated interior, with its shriveled placenta and dried-out pulp, leaving the pinkish-brown seeds without support. The cross-section of C1T3 also shows the lesion progression of the P. palmivora infection, penetrating through all the layers of the pericarp and spreading through more than half of the entire pod. Meanwhile, CTT1 was also opened for comparison as a control. The cacao pod was healthy – from its pericarp to its entire milky white pulp covering the seeds- with no signs of disease. All isolates caused lesions on unripe banana fruits. However, there is a difference in the severity of the lesions compared to its native host, cacao; the lesions were smaller in bananas than in cacao. On the third day, small reddish-brown to irregularly shaped reddish-brown lesions were observed around the wound of the infected banana. The average lesion diameter for an infected banana on the third day ranged from 8.78 mm to 10.89 mm. Moreover, the banana fruits infected with P. palmivora ripened on the 3rd day; earlier than the control. On the fifth day, most lesions for the isolates darkened in color, while the rest did not have any significant changes in appearance compared to the third day. The average lesion diameter for infected bananas on the fifth day widened, ranging from 11.89 mm to 13.88 mm. The lesions did not darken further on the seventh day, but the wound had shrunk. The average lesion diameter for infected bananas ranged from 9.89 mm to 13 mm. The lesion description of the banana fruits for the 3rd, 5th, and 7th day are elucidated in Supplementary Table 2. No lesions were observed for the negative control setups. After the measurement of lesions during the cross-infection analysis of P. palmivora on bananas on Day 7, select infected banana replicates and control banana replicates were sliced open horizontally to visualize the pathogen's infection inside the fruit. C1BT2 and C5BT1 were opened to reveal the cross-section of the infected banana fruit. Both replicates show the progression of the disease from inside the fruit through the spreading darkening areas with hints of green underneath the inoculation site. The green color is more pronounced on C5BT1, right below the dark lesion on the peel. CTBT1 was also opened as the control pod to compare with the infected pods. Several dark spots were seen on the skin of the banana – these were not considered as infection-induced lesions as the cross-section of CTBT1 revealed a healthy banana fruit with no lesion formation on the peel and no other signs of disease as seen in Fig. 8 . Like bananas, all isolates caused lesions in papaya but exhibited a lower degree of pathogenicity than cacao. On the third day, all isolates caused darkening on the inner circumference of the wound, with a few cases of brown lesions surrounding them. The average lesion diameter for an infected papaya on the third day ranged from 7.22 mm to 8.11 mm. On the fifth day, C1PT1 was observed with slight browning around its wound, C1PT2 showed browning around the wound while exhibiting reddish brown lesions on the upper and right side of the wound together with increased mycelia outside the wound, three of the isolates had an irregularly-shaped translucent brown lesion around their wound, some of the isolates exhibited slight darkening of the translucent brown lesion around their wound, C2PT3 was observed with minor translucent browning on the upper right side of the wound, C4PT3 slightly darkened with the manifestation of a translucent brown lesion and mycelia growing out the wound, C5PT1 exhibited irregularly-shaped translucent brown lesion around its wound with mycelia growing out of it, C5PT2 had increased areas with irregularly-shaped translucent to reddish brown lesion around the wound, C5PT3 showed darkening of its irregularly shaped brown lesions below the wound, and C2PT1 did not have any significant changes in terms of appearance compared to its third day. The average lesion diameter for an infected papaya on the fifth day ranged from 7.83 mm to 9.61 mm. On the seventh day, the majority of the isolates manifested a slight darkening of the brown lesions around their wound; both C1PT2 and C3PT1 had increased mycelia growing out of their wound, C2PT1 exhibited a slight browning on the right side of its wound, C2PT3 had translucent yellowish lesions around the wound, C4PT3 showed darkening of its brown lesion around its wound, and both C5PT2 and C5PT3 manifested a slight darkening of the irregularly shaped brown lesions below their wound. The average lesion diameter for an infected papaya on the seventh day ranged from 8 mm to 11.50 mm. The lesion description of the papaya fruits for the 3rd, 5th, and 7thday are elucidated in Supplementary Table 3. There were no significant changes in the appearance of the negative control nor the manifestation of wound shrinkage. After measuring lesions during the cross-infection analysis of P. palmivora on papaya fruits at Day 7, some infected papayas and control papaya replicates were sliced open horizontally to visualize the pathogen's infection inside the fruits. C1PT3 and C5PT2 were opened to show the fruits' cross-section, revealing mycelial formations and dark brown lesions formed at the inoculation sites with a light yellow-brownish area radiating from the circumference, as seen in Fig. 10 . Meanwhile, CTPT2 was also opened for comparison as the control fruit. Its cross-section revealed a healthy fruit without any darkening around the wounded site and no other observed signs of infection. Although only two isolates infected their host cacao, a higher degree of pathogenicity of P. palmivora is observed in cacao compared to banana and papaya. Similar findings were also observed in other cross-infection analyses in which the aggressiveness of the lesions was more advanced in their native hosts than their potential hosts [ 14 , 27 ]. P. palmivora presented a higher degree of pathogenicity in cacao, producing a lesion diameter ranging from 55 mm to 99 mm. In contrast, the isolates manifested a smaller lesion diameter of 9.89 mm to 13 mm in banana and 8 mm to 11.50 mm in papaya. Plants possess innate immunity, which pathogens such as P. palmivora must overcome. Phytophthora species adapted to impede the immunity of their specific plant hosts by utilizing effector proteins to manipulate and interfere with the plant's immune response [ 28 ]. Cross-infection studies indicate that the isolates of P. palmivora are less virulent in bananas than in cacao. The lesions observed in bananas were smaller and less aggressive than those found in cacao. This suggests a lower degree of pathogenicity in bananas compared to their native host, cacao. This is expected as the phylogenetic tree infers that the P. palmivora infecting cacao and the Phytophthora species infecting banana were separated into different lineages. However, the observed shrinking of the wound on the banana on the 7th day can be attributed to its faster ripening process compared to the control group. As the banana ripens, the peel "shrinks" as its water content decreases [ 29 ]. The lesions in papaya had the most minor and slowest disease progression compared to both cacao and banana. Similarly to bananas, the degree of pathogenicity varied in terms of severity. Papaya is a known host of P. palmivora [ 30 , 31 ]. Manifestation of the Phytophthora infection in papaya fruit is shown through off-white mycelium covering the wound and water-soaked lesions that turn brown as the disease progresses [ 32 , 33 ]. The slow progression of these symptoms may be attributed to the minute genetic differences of different P. palmivora strains that infect cacao and papaya. P. palmivora can inhibit plants' intrinsic immune response against pathogens through protease inhibitors [ 34 ]. However, the expression of these inhibitors varies from one plant host to another [ 35 , 36 , 37 , 38 ]. The varying degree of pathogenicity of the cacao isolates to the papaya fruits revealed differences in the isolated pathogen’s ability to propagate from its native host to other potential plant hosts. The phylogenetic tree also supports this, wherein the cacao isolates were quite distant from the P. palmivora strain, whose native host is the papaya. Discussion The main objective of this study was to conduct a cross-infection analysis of Phytophthora spp. on intercropped fruits isolated from cacao collected at Nagcarlan, Laguna, Philippines. While examining cacao pods that manifested black pod rot symptoms using Phytophthora spp.-selective agar, only one species was isolated, P. palmivora . Despite being the same species, the five selected P. palmivora isolates exhibited variations in their macroscopic and microscopic morphologies. However, molecular identification and phylogenetic analysis confirmed that all isolates belonged to the P. palmivora species regardless of morphological differences. The results showed that the isolates had a higher degree of pathogenicity in cacao, with mean lesion diameters ranging from 55 mm to 99 mm. In contrast, the isolates caused smaller mean lesion diameters of 9.89 mm to 13 mm in banana and 8 mm to 11.50 mm in papaya. The cross-infection studies revealed that the mean lesion diameter of bananas exhibited a marginally higher degree of pathogenicity when cross-infected with cacao isolates compared to the mean lesion diameter of papayas. Based on morphological and molecular analysis, Phytophthora palmivora is the only species isolated from the diseased cacao pods collected at the two intercropping farms at Nagcarlan, Laguna, Philippines. Cross-infection analysis suggested the potential for cross-infection of P. palmivora from its native host, cacao, to other plants, such as banana and papaya, when grown under the same environmental conditions. The lesions in papaya exhibited the most minor and slowest disease progression compared to cacao and banana. Meanwhile, bananas had a slightly higher degree of pathogenicity when compared to papaya. This variation in pathogenicity may be due to differences in the innate plant immune responses, indicating that bananas are more susceptible to infection by P. palmivora than papayas when intercropped with infected cacao plants. Isolating the same plant pathogen from various plant hosts is recommended. Future studies should also include more fruits for cross-infection analysis, considering their common intercropping with cacao. While pathogenicity testing is typically observed and quantified in the exocarp lesion, further studies should also explore its effects on the endocarp. Identifying these phytopathogens enables downstream applications such as discovering potential biocontrol agents. Additionally, sequencing effector proteins can enhance our understanding of the evolutionary relationships between P . palmivora isolates and their infection mechanisms across different plant hosts. Methods Collection of Cacao Pods Two intercropping farms in Nagcarlan, Laguna, Philippines, were the sites for the research’s survey and collection of Theobroma cacao pods. The two farms intercropped cacao with various plants, such as Artocarpus heterophyllus , Cocos nucifera , Carica papaya , and Musa sp . These farms were chosen as they reported experiencing an outbreak of a disease that causes black pod rot. Twenty-two cacao pods exhibiting black pod rot symptoms were collected through purposive sampling from the two farms. During the sample collection, the weather conditions experienced by the researchers were cloudy with rain showers and thunderstorms, which was confirmed by the Philippine Atmospheric, Geophysical, and Astronomical Services Administration’s regional weather forecast in Laguna for October 22, 2023 [ 39 ]. The exact coordinates of the cacao trees in which the cacao pod isolates were collected are listed in Table 4 . Table 4 Information on the cacao pod collection sites. This contains the date of collection, coordinates, altitude, and weather conditions during collection. Cacao Accession Number Plant ID Date of Collection Latitude Longitude Altitude Weather CP1 Theobroma cacao 10/22/2023 Pre-harvested Cloudy with intermittent rain showers CP2 10/22/2023 14.143404 121.378279 253 m CP3 10/22/2023 14.13692 121.385413 198 m CP4 10/22/2023 14.137016 121.385518 196 m CP5 10/22/2023 14.137067 121.385527 196 m CP6 10/22/2023 14.143439 121.378233 273 m CP7 10/22/2023 Pre-harvested CP8 10/22/2023 CP9 10/22/2023 14.143763 121.377948 253 m CP10 10/22/2023 14.143763 121.377911 253 m CP11 10/22/2023 14.143779 121.377791 249 m CP12 10/22/2023 14.143779 121.377791 249 CP13 10/22/2023 14.143756 121.377799 248 CP14 10/22/2023 14.143696 121.377904 249 CP15 10/22/2023 14.143734 121.377758 249 CP16 10/22/2023 14.143725 121.377677 249 CP17 10/22/2023 14.143725 121.377677 249 CP18 10/22/2023 14.143722 121.377727 249 CP19 10/22/2023 14.144206 121.377217 255 CP20 10/22/2023 14.137217 121.385637 257 CP21 10/22/2023 14.136738 121.385835 195 CP22 10/22/2023 Pre-harvested Isolation of Phytophthora spp. Pathogen samples were collected from the pods of Theobroma cacao L. exhibiting Phytophthora blight symptoms. The collected samples were brought to the PUP Mycology Laboratory for isolation. Before isolation, the samples were surface sterilized using 70% ethanol for one minute, rinsed three times with sterile distilled water, and dried on sterile tissue paper. Samples of 0.5 x 0.5 cm were excised from the cacao pericarp with partly necrotic and healthy tissues. After excision, these samples are embedded in a selective media of 20% clarified V8 agar buffered with 2 g/L calcium carbonate and amended with 100 ml/L nystatin, 200 mg/L ampicillin, and 100 mg/L rifampicin. Cultures were incubated at 25°C or room temperature in the dark for 2–3 days. Morphological Characterization of the Isolates Five Phytophthora isolates were used to characterize the culture and spore morphology based on their high percentage identity. Microscopic observation of cultural morphology was done using a stereomicroscope. Aseptic techniques were implemented while observing the mycelia and spores using a compound microscope. The tape method was used for observation, and the specimen was stained using lactophenol cotton blue. Molecular Identification and PCR Amplification of Samples of Phytophthora isolates were sentto the Department of Plant Pathology at the University of Florida for DNA extraction, amplification, and sequencing. V8 agar blocks from the colony edges of 5-day-old Phytophthora isolates containing mycelia were prepared in a sterile microcentrifuge tube and then shipped to the Department of Plant Pathology at the University of Florida, following the approval of the Material Transfer Agreement and permit from the Bureau of Plant Industry in the Philippines. Sequence and Phylogenetic Analysis Sequence and phylogenetic analysis were performed using two bioinformatics solutions, specifically NCBI Blast and Mega X. The processed sequences sent by the Department of Plant Phytopathology, University of Florida, were converted into FASTA format. The multiple and pairwise sequence alignment of the DNA nucleotides of the different Phytophthora species were rendered using MUSCLE via the MEGA program. The Pythium zingiberum strain (UOP389) from the NCBI GenBank is the chosen outgroup used in generating the phylogenetic tree. The best model generated using the MEGA program was chosen based on the lowest BIC scores. The phylogenetic tree was constructed using a bootstrap value of 1000 replicates [ 14 ]. Pathogenicity Test and Cross-Infection Test 5 Phytophthora isolates were used for the pathogenicity test on cacao and cross-infection tests on papaya and banana based on their high percentage identity. Pathogenicity and cross-infection tests were performed by artificial inoculation using a sterile 5 mm borer. Each isolate was tested in triplicate, with one sample unit per replicate. A 7-day-old Phytophthora culture, grown 5 days in the dark and 2 days in light, in 2 g/L calcium carbonate-buffered 20% V8 agar was used for the inoculum blocks or agar blocks. The healthy pods and fruits were surface sterilized using 70% ethanol for one minute, rinsed with sterile distilled water twice, and blot-dried with sterile tissue paper. Using the modified protocol of Solpot & Cumagun (2022), a 5 mm Phytophthora agar block was placed at the center of the artificially wounded cacao pods, papaya fruits, and banana fruits obtained in commercial markets [ 14 ]. The samples were incubated in the dark at 25°C. After three to seven days, successful inoculation was measured based on the extent of discoloration or lesions around the inoculation plugs by calculating the average lesion diameter in millimeters three times at different angles. Another triplicate control setup was prepared using the same 5 mm V8 agar blocks without the pathogen. Statistical Analysis The pathogenicity and cross-infection tests were conducted using a completely randomized design (CRD). The mean lesion diameter of all isolates underwent statistical analysis using the Statistical Tool for Agricultural Research (STAR), version 2.0.1 ( http://bbi.irri.org/products ), and data were analyzed using Tukey's Studentized Range (HSD) test to group isolates that are not statistically different at p < 0.05 [ 14 ]. Declarations Competing Interests The authors declare that the research was conducted with no competing interest. Funding Dr. Lourdes V. Alvarez received a Visiting Scholarship from Fulbright Philippines for the Molecular Analysis of the isolated samples at the University of Florida. Author Contribution Ricci Julia A. De Mesa, Myles David A. Florano, Jarvic Louis D. Suguitan, and Dr. Lourdes V. Alvarez equally contributed to this study. All authors reviewed the manuscript. Acknowledgement The authors would like to acknowledge Dr. Erica Goss and the Department of Plant Pathology, University of Florida, for aiding in this research. The authors appreciate the support from Fulbright Philippines for granting Dr. Lourdes V. Alvarez a Fulbright Scholarship grant at the University of Florida. Data Availability The datasets used and/or analysed during the current study is available from the corresponding author on reasonable request. References Zhang, D., Motilal, L. & Origin Dispersal, and Current Global Distribution of Cacao Genetic Diversity. Cacao Diseases: A History of Old Enemies and New Encounters (eds Meinhardt, L. W. & Bailey, B. A.) L. W. (Springer International Publishing, (2016). Kasteel, M., Ketelaar, T. & Govers, F. Fatal attraction: How Phytophthora zoospores find their host. Semin. Cell Dev. Biol. 148–149 , 13–21 (2023). Goss, E. M. et al. The Irish potato famine pathogen Phytophthora infestans originated in central Mexico rather than the Andes. 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First Report of Root and Basal Stem Rot Caused by Phytophthora cryptogea and P. inundata on Dwarf Banana in Italy. Am. Phytopathological Soc. 102 (3), 684. 10.1094/PDIS-08-17-1280-PDN (2018). Bentley, J. W., Boa, E. & Stonehouse, J. Neighbor Trees: Shade, Intercropping, and Cacao in Ecuador. Hum. Ecol. 32 (2), 241–270 (2004). Deemak, C., Lerslerwong, L., Nampila, S., Meetha, S. & Ayutthaya, S. I. N. Evaluation of Intercropped Papaya with Banana on Growth, Fruit, and Nutrient Status. Hortic. J. 92 (2), 142–150. 10.2503/hortj.QH-011 (2023). Tocafundo, F. et al. Direct and cross-pathogenicity of Phytophthora palmivora against cacao, papaya, and peach palm. Trop. Plant. Pathol. 46 , 664673. 10.1007/s40858-021-00464-y (2021). Solpot, T. C. & Cumagun, C. J. R. Phylogenetic analyses and cross-infection studies of Phytophthora species infecting cacao and durian in South‐Central Mindanao, Philippines. J. Phytopathol. 170 (1), 41–56 (2022). Dong, S. & Ma, W. How to win a tug-of-war: the adaptive evolution of Phytophthora effectors. Curr. Opin. Plant. Biol. 62 , 102027. 10.1016/j.pbi.2021.102027 (2021). Fraser, E. D. Social vulnerability and ecological fragility: building bridges between social and natural sciences using the Irish Potato Famine as a case study. Conservation Ecology 7 (2) (2003). Misman, N. et al. Host Range and Control Strategies of Phytophthora palmivora in Southeast Asia Perennial Crops. Pertanika J. Trop. Agric. Sci. 45 (4), 991–1019 (2022). Akrofi, A. Y., Amoaka-Atta, I., Assuah, M. & Asare, E. K. Black pod disease on cacao ( Theobroma cacao L.) in Ghana: Spread of Phytophthora megakarya and the role of economic plants in the disease epidemiology. Crop Prot. 72 , 66–75 (2015). Decloquement, J. et al. Phytophthora theobromicola sp. nov.: a new species causing black pod disease on cacao in Brazil. Front. Microbiol. 12 , 537399. 10.3389/fmicb.2021.537399 (2021). Kuswinati, T., Patandjengi, B. & Hardina, N. Melina, Morphological Variation and Molecular Characteristics of Phytophthora palmivora Isolates from Several Areas of Cocoa Plantations in South Sulawesi and Their Virulence on Sulawesi 2 Cocoa Clone. Jurnal Fit. Patologi Indonesia 19 (4) (2023). Alsultan, W. et al. Morphological, Pathogenic, and Molecular Characterization of Phytophthora palmivora Isolates Causing Black Pod Disease on Cocoa in Peninsular Malaysia. Int. J. Food Sci. Agric. 6 (2), 135–148 (2022). Kurbeti, I., Karaca, G., Aydogdu, M. & Sulu, G. Phytophthora Species Causing Root and Collar Rot of Pomegranate in Turkey. Eur. J. Plant Pathol. 157 , 485–496 (2020). Scanu, B. Phytophthora heterospora sp. nov., a new pseudoconidia-producing sister species of P. palmivora . J. Fungi . 7 (10), 870 (2021). Rodriguez-Polanco, E., Morales, J. G., Muñoz-Agudelo, M., Segura, J. D. & Carrero, M. L. Morphological, molecular and pathogenic characterization of Phytophthora palmivora isolates causing black pod rot of cacao in Colombia. Spanish J. Agricultural Research 18 (2) (2020). Muzuni, R. N. & Asniah, N. Y. Molecular Identification of Phytophthora sp. From Indonesian Cocoa Using Phylogenetic Analysis. Pak. J. Biol. Sci. 25 (3), 245–253 (2022). Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol. 35 , 1547–1549 (2018). Latifah, M., Kamaruzaman, S., Abidin, M. Z. & Nusaibah, S. A. Identification of Phytophthora spp. from perennial crops in Malaysia, its pathogenicity and cross-pathogenicity. Sains Malaysiana . 47 (5), 909–921 (2018). Perrine-Walker, F. Phytophthora palmivora –cocoa interaction. Journal Fungi . 6 (3), 167 (2020). Brat, P. et al. Post-harvest banana peel splitting as a function of relative humidity storage conditions. Acta Physiol. Plant. 38 , 1–14 (2016). de Oliveira, T. A. S., Blum, L. E. B., Duarte, E. A. A. & Luz, E. D. M. N. Control of Phytophthora palmivora on postharvest papaya with Trichoderma asperellum , T. virens, T. harzianum and T. longibrachiatum . Bioscience J. 34 (6), 1513–1521 (2018). Palmieri, D., Portillo, E., Sulbarán, Y., Guerra, M. & San-Blas, E. Biocontrol of Phytophthora root and stem rot disease in papaya ( Carica papaya ) plants by Photorhabdus, the symbiont bacterium of Heterorhabditis amazonensis . BioControl . 64 , 595–604 (2019). Vawdrey, L. L., Male, M. & Grice, K. R. Field and laboratory evaluation of fungicides for the control of Phytophthora fruit rot of papaya in far north Queensland, Australia. Crop Prot. 67 , 116–120 (2015). Singh, S. K., Dwivedi, M., Prasad, K., Sahu, S. R. & Chakravorty, S. Eco-Friendly Approach for Management of Postharvest Diseases of Tropical Fruit: Banana and Papaya. Management of Postharvest Diseases and Value Addition of Horticultural Crops (ed. Singh, D., Devappa, V., Jahagirdar, S., Gautam, H. R., and Aggarwal, R.) (Today and Tomorrow's Printers and Publishers, New Delhi, India, 2018). (2018). Gumtow, R., Wu, D., Uchida, J. & Tian, M. A Phytophthora palmivora extracellular cystatin-like protease inhibitor targets papain to contribute to virulence on papaya. Mol. Plant Microbe Interact. 31 (3), 363–373 (2018). Ekchaweng, K., Evangelisti, E., Schornack, S., Tian, M. & Churngchow, N. The plant defense and pathogen counterdefense mediated by Hevea brasiliensis serine protease HbSPA and Phytophthora palmivora extracellular protease inhibitor PpEPI10. PloS one . 12 (5), e0175795. 10.1371/journal.pone.0175795 (2017). Avila-Mendez, K., Rodrigo, Á., Araque, L. & Romero, H. M. Simultaneous transcriptome analysis of oil palm clones and Phytophthora palmivora reveals oil palm defense strategies. PloS one . 14 (9), e0222774. 10.1371/journal.pone.0222774 (2019). Pettongkhao, S., Navet, N., Schornack, S., Tian, M. & Churngchow, N. A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity. Sci. Rep. 10 (1), 2319. 10.1038/s41598-020-59007-1 (2020). Winters, N. P. et al. A combination of conserved and diverged responses underlies Theobroma cacao ’s defense response to Phytophthora palmivora . BMC Biol. 22 (1), 38 (2024). Philippine Atmospheric, Geophysical, and Astronomical Services Administration. Regional Weather Forecast for NCR-PRSD - October 22, 2023. PAGASA-DOST. (2023). https://pubfiles.pagasa.dost.gov.ph/ncrprsd/pf.pdf Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformationSR.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6390079","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453874192,"identity":"d7de15fb-389c-4b93-8169-6fb3be6cc5be","order_by":0,"name":"Ricci Julia De Mesa","email":"","orcid":"","institution":"Polytechnic University of the Philippines","correspondingAuthor":false,"prefix":"","firstName":"Ricci","middleName":"Julia","lastName":"De Mesa","suffix":""},{"id":453874193,"identity":"98a6dce9-c93e-4ec2-adb7-976920ec5ca4","order_by":1,"name":"Myles David Florano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFCCBCBik5OBcCqAmJm5gRgtxjwQzhmQFkYitDDAtDC2gUn8WnTbk59JPCgz4OGXPvzswcd5tdH87UAtPyq24dRiduaZmUTCOQMeyb40c8OZ247nzjjM2MDYc+Y2bi03EowNEtv+8BicYTCT5t12LLcBqIWZsQ2flvTPQC0GQC3s36R55xzLnU9YS47hA4gWHqAtDTW5GwhqOfOm8AHYLz08ZZIzjh3I3QjUchCvX46nbzj4o8xAjp+HfZvEh5q63HnnDx988KMCtxZ0cBhMHiBaPRDUkaJ4FIyCUTAKRggAAM3JWe6ojQYvAAAAAElFTkSuQmCC","orcid":"","institution":"Polytechnic University of the Philippines","correspondingAuthor":true,"prefix":"","firstName":"Myles","middleName":"David","lastName":"Florano","suffix":""},{"id":453874194,"identity":"c43b4dba-e024-453d-a4a0-f080e003fd02","order_by":2,"name":"Jarvic Louis Suguitan","email":"","orcid":"","institution":"Polytechnic University of the Philippines","correspondingAuthor":false,"prefix":"","firstName":"Jarvic","middleName":"Louis","lastName":"Suguitan","suffix":""},{"id":453874195,"identity":"d82b1bcf-1dca-4da0-bfca-e422c5412d01","order_by":3,"name":"Lourdes Alvarez","email":"","orcid":"","institution":"Polytechnic University of the Philippines","correspondingAuthor":false,"prefix":"","firstName":"Lourdes","middleName":"","lastName":"Alvarez","suffix":""}],"badges":[],"createdAt":"2025-04-07 04:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6390079/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6390079/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82389353,"identity":"ef645f72-4e0d-4aac-bc11-02f628989f0d","added_by":"auto","created_at":"2025-05-09 17:43:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":73588,"visible":true,"origin":"","legend":"\u003cp\u003eVarying colony morphology of the \u003cem\u003eP. palmivora\u003c/em\u003e isolates. Stellate striated pattern: CP1 (a), CP2 (b), CP3 (c). CP4 is cottony with a central rosette/chrysanthemum pattern and stellate radiate edge (d). CP5 with rosette/chrysanthemum pattern (e).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/386ac8a2b4eb42480ae5ef7c.jpg"},{"id":82389698,"identity":"8efce616-e0e0-4b96-acb9-6cf3b99eaed0","added_by":"auto","created_at":"2025-05-09 17:51:53","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66464,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of sporangia shape in \u003cem\u003eP. palmivora \u003c/em\u003eisolates. Different shapes include ovoid (a), globose (b), limoniform (c), and ellipsoid (d) shapes.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/532f1ef0bdb8e0dc24c890da.jpg"},{"id":82389699,"identity":"fa12aec3-0e37-4699-94b7-b3c1ffa853eb","added_by":"auto","created_at":"2025-05-09 17:51:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112300,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of chlamydospore shapes and location in \u003cem\u003eP. palmivora \u003c/em\u003eisolates. Different shapes include globose (a) and semiglobose (b). The locations of these chlamydospores may vary from being terminal (c), intercalary (d), or lateral (e).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/c9b794471f7b9e5899a28a6d.jpg"},{"id":82389358,"identity":"1a101991-f58d-482e-a540-9879d5d45741","added_by":"auto","created_at":"2025-05-09 17:43:53","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117254,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic Tree featuring the PUP \u003cem\u003ePhytophthora palmivora\u003c/em\u003e isolates alongside various \u003cem\u003ePhytophthora\u003c/em\u003especies of different origins and \u003cem\u003ePythium zingiberum\u003c/em\u003e. \u0026nbsp;The evolutionary history was inferred using the Maximum Likelihood method and Kimura 2-parameter model (Kimura, 1980). With this, a discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+\u003cem\u003eG\u003c/em\u003e, parameter = 0.2286)).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/05d0b7d798657ca5fb106d0a.jpg"},{"id":82390166,"identity":"b2f18f99-a25d-4d4b-838e-50471a34797c","added_by":"auto","created_at":"2025-05-09 17:59:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":155105,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenicity Test in Cacao Pods. The figure shows the gradual growth of lesions in the inoculated cacao pods along with the control for comparison.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/820ba43cb9590df28c8bca88.jpg"},{"id":82390457,"identity":"fbb244eb-8a1f-4f0b-95f1-f6d71a77fab7","added_by":"auto","created_at":"2025-05-09 18:07:53","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59502,"visible":true,"origin":"","legend":"\u003cp\u003eCross-section of Cacao Pods after Pathogenicity Testing. The figure shows and compares the cross-section of infected pods: C1T2 (a) and C1T3 (b), along with a control pod: CTT1 (c).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/3e47f39436cfb54b2116fc70.jpg"},{"id":82390167,"identity":"cacba8c1-dd63-4484-bd42-100a4cfe0620","added_by":"auto","created_at":"2025-05-09 17:59:53","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":82641,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenicity Test in Banana Fruits. The figure shows the gradual growth of lesions in the inoculated banana fruits and the control for comparison.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/1cafe2cb22b5b82e668b85c7.jpg"},{"id":82389704,"identity":"82c3589d-4c24-468d-b94c-5c2b42523e52","added_by":"auto","created_at":"2025-05-09 17:51:53","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":49823,"visible":true,"origin":"","legend":"\u003cp\u003eCross-section of Banana Fruits after Cross-Infection Analysis. The figure shows and compares the cross-section of two infected banana fruits: C1BT2 (a) and C5BT1 (b), along with a control fruit: CTBT1 (c).\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/44f8cde31f1855b227b17eeb.jpg"},{"id":82389365,"identity":"92b4e819-4111-421a-814f-b1da966b83d3","added_by":"auto","created_at":"2025-05-09 17:43:53","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":112954,"visible":true,"origin":"","legend":"\u003cp\u003ePathogenicity Test in Papaya Fruit. The figure shows the gradual growth of lesions in the inoculated papaya fruits and the control for comparison.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/a5bce88b884019c9bac6f48c.jpg"},{"id":82389700,"identity":"5ef0bcce-a6e2-4239-9551-110dc518cb95","added_by":"auto","created_at":"2025-05-09 17:51:53","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":35133,"visible":true,"origin":"","legend":"\u003cp\u003eCross-section of Papaya Fruits after Cross-infection Analysis. The figure shows and compares the cross-section of infected papaya fruits C1PT3 (a) and C5PT2 (b), along with a control fruit, CTPT2 (c).\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/6af8fbeaecad44a5c851b960.jpg"},{"id":87179193,"identity":"f51323c4-7fae-47f2-9af5-4204fff573bc","added_by":"auto","created_at":"2025-07-21 09:23:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1884156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/95d54749-5c7c-4fa6-9a22-582b15fe1172.pdf"},{"id":82389355,"identity":"606b3937-73fb-4147-9983-d5e861e9b50c","added_by":"auto","created_at":"2025-05-09 17:43:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":12456,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationSR.docx","url":"https://assets-eu.researchsquare.com/files/rs-6390079/v1/781d9a26ddd816dd0c33414a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cross-Infection Analysis of Phytophthora spp. Isolated from Cacao (Theobroma cacao L.) Pods at Nagcarlan, Laguna, Philippines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCacao is the raw material for the world-renowned delicacy known as chocolate. Despite a high demand for cacao, its production remains low [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among many factors, phytopathogens contribute significantly to the low production of cacao.\u003c/p\u003e \u003cp\u003e\u003cem\u003ePhytophthora\u003c/em\u003e spp. are pathogenic oomycetes that target numerous plant species. They infect plant species through their zoospores, which are motile, asexual spores. Using their flagella, these can move according to physical, electrical, and chemical cues, allowing them to navigate to sites within the plant that they can infect, including the stomata, wound sites, and roots [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This mechanism of infection proved to be incredibly effective, with \u003cem\u003ePhytophthora\u003c/em\u003e species having already caused numerous historical social crises during the 1800s, including the European Famine, the Great Famine of Ireland, and the Highland Famines. The species that caused these famines was identified as \u003cem\u003eP. infestans\u003c/em\u003e, which originated in Central Mexico and was then unknowingly introduced to Europe, where it wrought devastation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite causing such ravaging events in history nearly 200 years ago, the full extent of the genus \u003cem\u003ePhytophthora\u003c/em\u003e is yet to be thoroughly studied, with research suggesting an estimated number of about 500 species of \u003cem\u003ePhytophthora\u003c/em\u003e exist all around the world but are yet to be identified [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Having unknown species of \u003cem\u003ePhytophthora\u003c/em\u003e out in the wild can have new implications, such as a far more extensive host range and varying degrees of pathogenicity compared to what is currently known [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This uncertainty, along with the dangerous precedent set two centuries ago for the ruin it can bring, is indeed enough to raise concerns, not only for cacao but also for entire farms that implement intercropping cacao with other potential hosts.\u003c/p\u003e \u003cp\u003eNumerous studies have been conducted and published to identify unknown species of Phytophthora in the wild and understand their capacity to cause damage to different plant hosts. According to the United States Department of Agriculture\u0026rsquo;s research headed by Brasier in 2009, novel \u003cem\u003ePhytophthora\u003c/em\u003e species are constantly being discovered worldwide, each adapting to different plant species as their hosts [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003ePhytophthora\u003c/em\u003e spp. are known for their diverse host range and highly virulent characteristics. Aside from cacao, other fruits are also devastated by various \u003cem\u003ePhytophthora\u003c/em\u003e spp. Since cacao is usually intercropped with other plants, the chances of passing on \u003cem\u003ePhytophthora\u003c/em\u003e spp. from one plant species to another is high. Upon an interview with the farmers from Nagcarlan, Laguna, Philippines, they revealed their usual intercrops with cacao, which include rambutan (\u003cem\u003eNephelium lappaceum\u003c/em\u003e L. var. \u003cem\u003elappaceum\u003c/em\u003e), banana (\u003cem\u003eMusa\u003c/em\u003e spp.), coconut (\u003cem\u003eCocos nucifera\u003c/em\u003e L.), lanzones (\u003cem\u003eLansium domesticum\u003c/em\u003e), jackfruit (\u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e), avocado (\u003cem\u003ePersea americana\u003c/em\u003e), and durian (\u003cem\u003eDurio zibethinus\u003c/em\u003e L.). This study focuses on bananas and papayas for convenience since they are of the optimum size and not seasonal.\u003c/p\u003e \u003cp\u003eTan (2021) wrote that cacao and bananas are promising intercropping duos because they use the same fertilizer and do not attract the same types of pests [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previously, \u003cem\u003ePhytophthora\u003c/em\u003e spp. was not viewed as a threat in bananas (Stover 1972) until Thompson (1981) recorded the presence of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003enicotinae\u003c/em\u003e B. de Haan var. \u003cem\u003eparasitca\u003c/em\u003e (Dastur) Waterh. in a devastating Phytophthora finger rot in South Africa [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. He described the symptoms as brown-margined with a firm black lesion on the peel and pulp, sometimes covered by a superficial, felty, white mycelium. Moreover, the possibility that bananas could be an alternate host for \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epalmivora\u003c/em\u003e in cacao plantations was also reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In recent reports, \u003cem\u003eP\u003c/em\u003e. \u003cem\u003ecryptogea\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003einundata\u003c/em\u003e causing root and basal stem rot were detected on dwarf bananas in Italy [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother good intercrop for cacao and banana is papaya [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, papaya is a known susceptible host to \u003cem\u003ePhytophthora\u003c/em\u003e. The known species that cause \u003cem\u003ePhytophthora\u003c/em\u003e blight in papaya include \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epalmivora\u003c/em\u003e and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003enicotinae\u003c/em\u003e. The symptoms of \u003cem\u003ePhytophthora\u003c/em\u003e blight in papaya include water-soaked lesions with milky latex, fruit rot with white mycelium growth, stem cankers, and dark, rotten roots.\u003c/p\u003e \u003cp\u003eResearch conducted by Tocafundo et al. (2021) studied the direct and cross-infection of \u003cem\u003eP. palmivora\u003c/em\u003e on cacao intercropped with papaya and peach palm [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It was found that while the derived \u003cem\u003eP. palmivora\u003c/em\u003e isolates can infect other plant species, the specificity of the \u003cem\u003ePhytophthora\u003c/em\u003e species meant that they were far more aggressive on their original host. While this means there would be less damage inflicted than anticipated in intercrop systems, it still exists. Moreover, given enough time to evolve according to their new host, the \u003cem\u003ePhytophthora\u003c/em\u003e species may become just as pathogenic or even more.\u003c/p\u003e \u003cp\u003eLocal research was also conducted in South-Central Mindanao, where \u003cem\u003ePhytophthora\u003c/em\u003e spp. isolated from infected cacao and durian trees were cross-infected with potential hosts, rubber and coconut. These four were used as these were often grown in intercrop systems in that part of Mindanao. The research found that \u003cem\u003eP. palmivora\u003c/em\u003e was pathogenic to all the tested hosts [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have made promising progress in combating Phytophthora infections in genetics. As zoospores infect hosts via taxis, researchers have avoided host recognition by silencing target genes, such as the \u003cem\u003eAvr\u003c/em\u003e gene [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While this breakthrough was successful in the laboratory, it may take some time and more research before this method can be deployed in real-world applications. Furthermore, as the fear of cross-infection caused by \u003cem\u003ePhytophthora\u003c/em\u003e within intercrop systems sets in, it may be misconstrued that the solution to the problem is monocropping, but it is not. As mentioned earlier, the Great Famine of Ireland was partially caused by the country\u0026rsquo;s dependence on potato and tomato monocropping [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith this situation in hand, this research investigated the possible cross-infection of \u003cem\u003ePhytophthora\u003c/em\u003e species isolated from cacao collected within the local geographic region, Nagcarlan, Laguna, Philippines, to papaya and banana. This serves as a stepping stone in understanding these pathogenic species even more and learning which plants are in danger of infection within intercrop systems. Specifically, this study aims to isolate \u003cem\u003ePhytophthora\u003c/em\u003e spp. from diseased cacao at Nagcarlan, Laguna, Philippines, and identify these through polyphasic methods. After successful isolation, the extent of cross-infection of these \u003cem\u003ePhytophthora\u003c/em\u003e spp. was analyzed in banana and papaya.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe collection conditions of the cacao pods were considered factors in successfully isolating the samples. During the cacao pod collection on October 22, 2023, a heavy downpour of rain ensued throughout the collection period at the collection sites in Nagcarlan, Laguna. From previous studies, it has been observed in other hot-humid countries in Southeast Asia that \u003cem\u003eP. palmivora\u003c/em\u003e infects faster and spreads more during the wet seasons as the water brought on by the rain allows the flagellated zoospores of the \u003cem\u003eP. palmivora\u003c/em\u003e to swim and infect nearby hosts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, the geographical structure of the two farms, while different from each other, was still a considerable factor in the success of isolating the samples. The steep slopes of the first farm help propagate the \u003cem\u003eP. palmivora\u003c/em\u003e spores as water-carrying zoospores from infected plants at the top of the hill flow downward to the other plants at the bottom, promoting infection. The second farm has a flat and open field where water could accumulate on its surface, spreading disease around it [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhytophthora palmivora\u003c/b\u003e \u003cb\u003eMorphology\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOnly five (5) isolates were described morphologically due to their high percentage identity. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the macroscopic and microscopic morphology of \u003cem\u003eP. palmivora\u003c/em\u003e isolates in V8 agar. Overall, there were three major colony patterns observed: (1) stellate striated pattern (n\u0026thinsp;=\u0026thinsp;3), (2) cottony with central rosette/chrysanthemum pattern and stellate radiate edge (n\u0026thinsp;=\u0026thinsp;1), and a consistent (3) rosette/chrysanthemum pattern (n\u0026thinsp;=\u0026thinsp;1). These patterns are congruent with the morphology of the isolated \u003cem\u003eP. palmivora\u003c/em\u003e of Solpot and Cumagun (2022), whose isolation is done in the same country but in a different region [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The stellate pattern is typical in all \u003cem\u003ePhytophthora\u003c/em\u003e species and not just \u003cem\u003eP. palmivora\u003c/em\u003e as seen in the \u003cem\u003eP. megakarya\u003c/em\u003e isolates of Akrofi et al. (2015) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Moreover, the rosette or chrysanthemum growth pattern in \u003cem\u003eP. palmivora\u003c/em\u003e was also reported by Solpot and Cumagun (2022) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Still, this unique growth pattern can also be seen in the novel species of \u003cem\u003eP. theobromicola\u003c/em\u003e sp. nov. reported by Decloquement et al. (2021) in Brazil [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Based on its colony growth, the CP1 colony can reach 35\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm in 2 days, the CP2 colony can reach 36\u0026thinsp;\u0026plusmn;\u0026thinsp;1 mm in 2 days, the CP3 and CP5 colonies can reach 40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm in 2 days, and the CP4 colony can reach 38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 mm in 2 days. Combined, \u003cem\u003eP. palmivora\u003c/em\u003e isolates exhibited a growth rate of 38\u0026thinsp;\u0026plusmn;\u0026thinsp;2 mm in 2 days.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of colony pattern, sporangia morphology, and chlamydospores morphology of the isolated \u003cem\u003eP. palmivora.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003cp\u003eCode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIdentity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eColony Pattern\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSporangia Morphology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eChlamydospores Morphology\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP.\u0026nbsp;\u0026nbsp;palmivora\u003c/em\u003e\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estellate striated pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShape: ovoid, globose, limoniform\u003c/p\u003e \u003cp\u003eLength: 37\u0026thinsp;\u0026plusmn;\u0026thinsp;5 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShape: globose\u003c/p\u003e \u003cp\u003eLocation: terminal\u003c/p\u003e \u003cp\u003eLength: 38\u0026thinsp;\u0026plusmn;\u0026thinsp;4 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP.\u0026nbsp;\u0026nbsp;palmivora\u003c/em\u003e\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estellate striated pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShape: ovoid, globose \u003c/p\u003e \u003cp\u003eLength: 24\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShape: globose\u003c/p\u003e \u003cp\u003eLocation: terminal, intercalary, lateral \u003c/p\u003e \u003cp\u003eLength: 29\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP.\u0026nbsp;\u0026nbsp;palmivora\u003c/em\u003e\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003estellate striated pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShape: ovoid, globose \u003c/p\u003e \u003cp\u003eLength: 28\u0026thinsp;\u0026plusmn;\u0026thinsp;9 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShape: globose \u003c/p\u003e \u003cp\u003eLocation: terminal, intercalary\u003c/p\u003e \u003cp\u003eLength: 30\u0026thinsp;\u0026plusmn;\u0026thinsp;6 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP.\u0026nbsp;\u0026nbsp;palmivora\u003c/em\u003e\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecottony with central rosette / chrysanthemum pattern and stellate radiate edge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShape: ovoid, globose\u003c/p\u003e \u003cp\u003eLength: 30\u0026thinsp;\u0026plusmn;\u0026thinsp;7 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShape: globose, semi globose\u003c/p\u003e \u003cp\u003eLocation: terminal\u003c/p\u003e \u003cp\u003eLength: 28\u0026thinsp;\u0026plusmn;\u0026thinsp;5 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP.\u0026nbsp;\u0026nbsp;palmivora\u003c/em\u003e\u0026nbsp;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003erosette / chrysanthemum pattern\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShape: ovoid, globose, ellipsoid, limoniform\u003c/p\u003e \u003cp\u003eLength: 28\u0026thinsp;\u0026plusmn;\u0026thinsp;7 mm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShape: globose\u003c/p\u003e \u003cp\u003eLocation: terminal, intercalary\u003c/p\u003e \u003cp\u003eLength: 30\u0026thinsp;\u0026plusmn;\u0026thinsp;4 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThere was also variation in the sporangia shape, but all isolates uniformly presented common shapes, which are ovoid and globose with evident papillae. Kuswinati et al. (2023) reported a varying papilla of \u003cem\u003ePhytophthora\u003c/em\u003e sporangia, which can be papilated or non-papilated [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the ovoid sporangia shape was the most abundant of all isolates. This is also true for the \u003cem\u003eP. palmivora\u003c/em\u003e isolated by Alsultan et al. (2022) in Malaysia [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, CP1 and CP5 showed a limoniform sporangia shape, and CP5 showed a unique ellipsoid sporangia shape. The lengths of all sporangia vary even within the same isolate.\u003c/p\u003e \u003cp\u003eIn terms of chlamydospores, all isolates showed uniform terminal globose chlamydospores, while some isolates showed intercalary and lateral chlamydospores. Some chlamydospores showed a semi-globose shape. All these morphological characteristics of chlamydospores are common in \u003cem\u003eP. palmivora\u003c/em\u003e and other \u003cem\u003ePhytophthora\u003c/em\u003e spp., as reported by Kurbeti et al. (2020), Scanu et al. (2021), Alsultan et al. (2022), and Kuswinati et al. (2023) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDifferent \u003cem\u003ePhytophthora\u003c/em\u003e spp. have similar morphological characteristics, while the same species of \u003cem\u003ePhytophthora\u003c/em\u003e may show varying morphological characteristics. Thus, morphological characterization is only helpful in identifying \u003cem\u003ePhytophthora\u003c/em\u003e at the genus level. Several studies also report variations in colony morphology and microscopic morphology between the same species of \u003cem\u003eP. palmivora\u003c/em\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMolecular and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eFollowing the isolation of the samples from the cacao pods, molecular identification of the samples was started simultaneously with the morphological analysis. The samples were sent to the Plant Pathology Department of the University of Florida for DNA sequencing. Out of the twenty-two isolates sent, twenty-one were revived successfully following the delivery of the samples overseas, which were then sequenced. The resulting sequences sent back were cross-checked with sequences from the NCBI GenBank for identification. It was found that all twenty-one isolates were identified as \u003cem\u003ePhytophthora palmivora\u003c/em\u003e. From those, five isolates that presented with high percent identities were chosen as representatives for this study, which are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The selected five isolates, CP1, CP2, CP3, CP4, and CP5, all matched with \u003cem\u003eP. palmivora\u003c/em\u003e isolate UHO-1 (MT644188.1) on NCBI\u0026rsquo;s GenBank [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] with 99.39%, 99.39%, 100%, 99.85%, and 99.23% percent identities, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecular identification of the five isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate Code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIdentification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercent Identity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccession Number\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003ePhytophthora palmivora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.39%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eMT644188.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.39%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.85%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.23%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese sequences then underwent phylogenetic analysis with the use of MEGA X [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], along with additional reference sequences for comparison retrieved from NCBI\u0026rsquo;s GenBank, including ten reference sequences of \u003cem\u003eP. palmivora\u003c/em\u003e isolated from \u003cem\u003eTheobroma cacao\u003c/em\u003e (isolates 227, TARS1, I320, CNC-P8, UHO-1, PPM1, H12, Co-17, CPR22, and CBS179.26), two reference sequences of \u003cem\u003eP. palmivora\u003c/em\u003e isolated from \u003cem\u003eCarica papay\u003c/em\u003ea (isolates CHA134 and BRIP 40506a), five reference sequences of \u003cem\u003ePhytophthora sp.\u003c/em\u003e from Type Material: \u003cem\u003eP. litoralis, P. multivora, P. plurivora, P. capsici\u003c/em\u003e, and \u003cem\u003eP. tropicalis\u003c/em\u003e, two reference sequences of \u003cem\u003ePhytophthora sp.\u003c/em\u003e isolated from \u003cem\u003eMusa sp\u003c/em\u003e. (\u003cem\u003eP. cryptogea\u003c/em\u003e PHBan1 and \u003cem\u003eP. inundata\u003c/em\u003e PHBan3), and one reference sequence of \u003cem\u003ePythium zingiberum\u003c/em\u003e (isolate UOP389) as the outgroup. The resulting Phylogenetic Tree is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAll the formed clades in the Phylogenetic Tree are strongly supported, with bootstrap values ranging between 98\u0026ndash;100%. Starting from the bottom of the tree, the \u003cem\u003ePhytophthora sp.\u003c/em\u003e clade can be seen diverging from the \u003cem\u003ePythium zingiberum\u003c/em\u003e by a large margin, which is to be expected from the outgroup. Among the referenced Type Material \u003cem\u003ePhytophthora sp\u003c/em\u003e. isolates, \u003cem\u003eP. tropicalis\u003c/em\u003e and \u003cem\u003eP. capsici\u003c/em\u003e were the first to diverge away from the group, which was then followed by the clade containing \u003cem\u003eP. plurivora\u003c/em\u003e and \u003cem\u003eP. multivora\u003c/em\u003e. The remaining isolates were then divided into two groups; the first comprised of \u003cem\u003eP. cryptogea\u003c/em\u003e and a minor clade containing the \u003cem\u003eP. inundata\u003c/em\u003e and \u003cem\u003eP. litoralis\u003c/em\u003e. The second clade comprised the \u003cem\u003eP. palmivora\u003c/em\u003e isolates, including the PUP Cacao Isolates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePathogenicity Test and Cross-Infection Test\u003c/h3\u003e\n\u003cp\u003eThe results of the pathogenicity and cross-infection tests of the five isolates of \u003cem\u003ePhytophthora palmivora\u003c/em\u003e from infected cacao are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Two isolates infected unripe cacao pods with varying degrees of severity. Brown-reddish lesions were first observed on the third day of C1T2 with an average diameter of 50mm. On the fifth day, all replicates of C1 varied in the severity of infection, showing brown-reddish to dark brown lesions with the appearance of mycelia and spores over them. Concurrently, dark brown lesions were observed around the wound of C3T2. The average lesion diameter for infected cacao on the fifth day ranged from 29 mm to 61.78 mm. On the seventh day, all replicates of C1 showed widened and darkened brown lesions with increased manifestation of mycelia and spores. At the same time, two replicates of C3 showed varying degrees of infection; C3T1 exhibited an irregular brown lesion near the wound, while C3T2 presented a spread-out dark brown lesion with an increased amount of mycelia and spores. The average lesion diameter for infected cacao on the seventh day ranged from 55 mm to 99 mm. The lesion description of the cacao pods for the third, fifth, and seventh-day are elucidated in Supplementary Table\u0026nbsp;1. None of the negative control setups developed lesions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean lesion diameter of \u003cem\u003ePhytophthora palmivora\u003c/em\u003e isolates on cacao, banana, and papaya after 7 days of inoculation. \u003cem\u003e*Means within the same column followed by a common letter are not significantly different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 using Tukey\u0026rsquo;s Studentized Range (HSD) test. The mean of lesions was calculated with three independent replicates per sample unit.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate Code\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInoculum source\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCacao\u003c/p\u003e \u003cp\u003e(7th day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBanana\u003c/p\u003e \u003cp\u003e(7th day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePapaya\u003c/p\u003e \u003cp\u003e(7th day)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cem\u003ePhytophthora palmivora\u003c/em\u003e (cacao)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.11\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.89\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.78\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.67\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.33\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.67\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.5\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.22\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.78\u003csub\u003eab\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.5\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003csub\u003eb\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFollowing the measurement of the lesions on Day 7, the diseased cacao pods were sliced open horizontally to visualize the cross-section of the cacao pods after being infected by \u003cem\u003eP. palmivora\u003c/em\u003e. C1T2 was opened to reveal some mild shrinkage of the cacao\u0026rsquo;s pulp and browning of the placenta. With this cross-section, the spread of the \u003cem\u003eP. palmivora\u003c/em\u003e-induced lesion on the 7th day is also visualized, and it was observed to have encompassed the entire cacao pod\u0026rsquo;s pericarp. C1T3 was also opened to reveal an entirely devastated interior, with its shriveled placenta and dried-out pulp, leaving the pinkish-brown seeds without support. The cross-section of C1T3 also shows the lesion progression of the \u003cem\u003eP. palmivora\u003c/em\u003e infection, penetrating through all the layers of the pericarp and spreading through more than half of the entire pod. Meanwhile, CTT1 was also opened for comparison as a control. The cacao pod was healthy \u0026ndash; from its pericarp to its entire milky white pulp covering the seeds- with no signs of disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll isolates caused lesions on unripe banana fruits. However, there is a difference in the severity of the lesions compared to its native host, cacao; the lesions were smaller in bananas than in cacao. On the third day, small reddish-brown to irregularly shaped reddish-brown lesions were observed around the wound of the infected banana. The average lesion diameter for an infected banana on the third day ranged from 8.78 mm to 10.89 mm. Moreover, the banana fruits infected with \u003cem\u003eP. palmivora\u003c/em\u003e ripened on the 3rd day; earlier than the control. On the fifth day, most lesions for the isolates darkened in color, while the rest did not have any significant changes in appearance compared to the third day. The average lesion diameter for infected bananas on the fifth day widened, ranging from 11.89 mm to 13.88 mm. The lesions did not darken further on the seventh day, but the wound had shrunk. The average lesion diameter for infected bananas ranged from 9.89 mm to 13 mm. The lesion description of the banana fruits for the 3rd, 5th, and 7th day are elucidated in Supplementary Table\u0026nbsp;2. No lesions were observed for the negative control setups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter the measurement of lesions during the cross-infection analysis of \u003cem\u003eP. palmivora\u003c/em\u003e on bananas on Day 7, select infected banana replicates and control banana replicates were sliced open horizontally to visualize the pathogen's infection inside the fruit. C1BT2 and C5BT1 were opened to reveal the cross-section of the infected banana fruit. Both replicates show the progression of the disease from inside the fruit through the spreading darkening areas with hints of green underneath the inoculation site. The green color is more pronounced on C5BT1, right below the dark lesion on the peel. CTBT1 was also opened as the control pod to compare with the infected pods. Several dark spots were seen on the skin of the banana \u0026ndash; these were not considered as infection-induced lesions as the cross-section of CTBT1 revealed a healthy banana fruit with no lesion formation on the peel and no other signs of disease as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLike bananas, all isolates caused lesions in papaya but exhibited a lower degree of pathogenicity than cacao. On the third day, all isolates caused darkening on the inner circumference of the wound, with a few cases of brown lesions surrounding them. The average lesion diameter for an infected papaya on the third day ranged from 7.22 mm to 8.11 mm. On the fifth day, C1PT1 was observed with slight browning around its wound, C1PT2 showed browning around the wound while exhibiting reddish brown lesions on the upper and right side of the wound together with increased mycelia outside the wound, three of the isolates had an irregularly-shaped translucent brown lesion around their wound, some of the isolates exhibited slight darkening of the translucent brown lesion around their wound, C2PT3 was observed with minor translucent browning on the upper right side of the wound, C4PT3 slightly darkened with the manifestation of a translucent brown lesion and mycelia growing out the wound, C5PT1 exhibited irregularly-shaped translucent brown lesion around its wound with mycelia growing out of it, C5PT2 had increased areas with irregularly-shaped translucent to reddish brown lesion around the wound, C5PT3 showed darkening of its irregularly shaped brown lesions below the wound, and C2PT1 did not have any significant changes in terms of appearance compared to its third day. The average lesion diameter for an infected papaya on the fifth day ranged from 7.83 mm to 9.61 mm. On the seventh day, the majority of the isolates manifested a slight darkening of the brown lesions around their wound; both C1PT2 and C3PT1 had increased mycelia growing out of their wound, C2PT1 exhibited a slight browning on the right side of its wound, C2PT3 had translucent yellowish lesions around the wound, C4PT3 showed darkening of its brown lesion around its wound, and both C5PT2 and C5PT3 manifested a slight darkening of the irregularly shaped brown lesions below their wound. The average lesion diameter for an infected papaya on the seventh day ranged from 8 mm to 11.50 mm. The lesion description of the papaya fruits for the 3rd, 5th, and 7thday are elucidated in Supplementary Table\u0026nbsp;3. There were no significant changes in the appearance of the negative control nor the manifestation of wound shrinkage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter measuring lesions during the cross-infection analysis of \u003cem\u003eP. palmivora\u003c/em\u003e on papaya fruits at Day 7, some infected papayas and control papaya replicates were sliced open horizontally to visualize the pathogen's infection inside the fruits. C1PT3 and C5PT2 were opened to show the fruits' cross-section, revealing mycelial formations and dark brown lesions formed at the inoculation sites with a light yellow-brownish area radiating from the circumference, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Meanwhile, CTPT2 was also opened for comparison as the control fruit. Its cross-section revealed a healthy fruit without any darkening around the wounded site and no other observed signs of infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough only two isolates infected their host cacao, a higher degree of pathogenicity of \u003cem\u003eP. palmivora\u003c/em\u003e is observed in cacao compared to banana and papaya. Similar findings were also observed in other cross-infection analyses in which the aggressiveness of the lesions was more advanced in their native hosts than their potential hosts [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. \u003cem\u003eP. palmivora\u003c/em\u003e presented a higher degree of pathogenicity in cacao, producing a lesion diameter ranging from 55 mm to 99 mm. In contrast, the isolates manifested a smaller lesion diameter of 9.89 mm to 13 mm in banana and 8 mm to 11.50 mm in papaya.\u003c/p\u003e \u003cp\u003ePlants possess innate immunity, which pathogens such as \u003cem\u003eP. palmivora\u003c/em\u003e must overcome. Phytophthora species adapted to impede the immunity of their specific plant hosts by utilizing effector proteins to manipulate and interfere with the plant's immune response [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Cross-infection studies indicate that the isolates of \u003cem\u003eP. palmivora\u003c/em\u003e are less virulent in bananas than in cacao. The lesions observed in bananas were smaller and less aggressive than those found in cacao. This suggests a lower degree of pathogenicity in bananas compared to their native host, cacao. This is expected as the phylogenetic tree infers that the \u003cem\u003eP. palmivora\u003c/em\u003e infecting cacao and the \u003cem\u003ePhytophthora\u003c/em\u003e species infecting banana were separated into different lineages.\u003c/p\u003e \u003cp\u003eHowever, the observed shrinking of the wound on the banana on the 7th day can be attributed to its faster ripening process compared to the control group. As the banana ripens, the peel \"shrinks\" as its water content decreases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe lesions in papaya had the most minor and slowest disease progression compared to both cacao and banana. Similarly to bananas, the degree of pathogenicity varied in terms of severity. Papaya is a known host of \u003cem\u003eP. palmivora\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Manifestation of the Phytophthora infection in papaya fruit is shown through off-white mycelium covering the wound and water-soaked lesions that turn brown as the disease progresses [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The slow progression of these symptoms may be attributed to the minute genetic differences of different \u003cem\u003eP. palmivora\u003c/em\u003e strains that infect cacao and papaya. \u003cem\u003eP. palmivora\u003c/em\u003e can inhibit plants' intrinsic immune response against pathogens through protease inhibitors [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, the expression of these inhibitors varies from one plant host to another [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The varying degree of pathogenicity of the cacao isolates to the papaya fruits revealed differences in the isolated pathogen\u0026rsquo;s ability to propagate from its native host to other potential plant hosts. The phylogenetic tree also supports this, wherein the cacao isolates were quite distant from the \u003cem\u003eP. palmivora\u003c/em\u003e strain, whose native host is the papaya.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main objective of this study was to conduct a cross-infection analysis of \u003cem\u003ePhytophthora\u003c/em\u003e spp. on intercropped fruits isolated from cacao collected at Nagcarlan, Laguna, Philippines. While examining cacao pods that manifested black pod rot symptoms using \u003cem\u003ePhytophthora\u003c/em\u003e spp.-selective agar, only one species was isolated, \u003cem\u003eP. palmivora\u003c/em\u003e. Despite being the same species, the five selected \u003cem\u003eP. palmivora\u003c/em\u003e isolates exhibited variations in their macroscopic and microscopic morphologies. However, molecular identification and phylogenetic analysis confirmed that all isolates belonged to the P. palmivora species regardless of morphological differences. The results showed that the isolates had a higher degree of pathogenicity in cacao, with mean lesion diameters ranging from 55 mm to 99 mm. In contrast, the isolates caused smaller mean lesion diameters of 9.89 mm to 13 mm in banana and 8 mm to 11.50 mm in papaya. The cross-infection studies revealed that the mean lesion diameter of bananas exhibited a marginally higher degree of pathogenicity when cross-infected with cacao isolates compared to the mean lesion diameter of papayas.\u003c/p\u003e \u003cp\u003eBased on morphological and molecular analysis, \u003cem\u003ePhytophthora palmivora\u003c/em\u003e is the only species isolated from the diseased cacao pods collected at the two intercropping farms at Nagcarlan, Laguna, Philippines. Cross-infection analysis suggested the potential for cross-infection of \u003cem\u003eP. palmivora\u003c/em\u003e from its native host, cacao, to other plants, such as banana and papaya, when grown under the same environmental conditions. The lesions in papaya exhibited the most minor and slowest disease progression compared to cacao and banana. Meanwhile, bananas had a slightly higher degree of pathogenicity when compared to papaya. This variation in pathogenicity may be due to differences in the innate plant immune responses, indicating that bananas are more susceptible to infection by \u003cem\u003eP. palmivora\u003c/em\u003e than papayas when intercropped with infected cacao plants.\u003c/p\u003e \u003cp\u003eIsolating the same plant pathogen from various plant hosts is recommended. Future studies should also include more fruits for cross-infection analysis, considering their common intercropping with cacao. While pathogenicity testing is typically observed and quantified in the exocarp lesion, further studies should also explore its effects on the endocarp. Identifying these phytopathogens enables downstream applications such as discovering potential biocontrol agents. Additionally, sequencing effector proteins can enhance our understanding of the evolutionary relationships between \u003cem\u003eP\u003c/em\u003e. \u003cem\u003epalmivora\u003c/em\u003e isolates and their infection mechanisms across different plant hosts.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCollection of Cacao Pods\u003c/h2\u003e \u003cp\u003eTwo intercropping farms in Nagcarlan, Laguna, Philippines, were the sites for the research\u0026rsquo;s survey and collection of \u003cem\u003eTheobroma cacao\u003c/em\u003e pods. The two farms intercropped cacao with various plants, such as \u003cem\u003eArtocarpus heterophyllus\u003c/em\u003e, \u003cem\u003eCocos nucifera\u003c/em\u003e, \u003cem\u003eCarica papaya\u003c/em\u003e, and \u003cem\u003eMusa sp\u003c/em\u003e. These farms were chosen as they reported experiencing an outbreak of a disease that causes black pod rot. Twenty-two cacao pods exhibiting black pod rot symptoms were collected through purposive sampling from the two farms. During the sample collection, the weather conditions experienced by the researchers were cloudy with rain showers and thunderstorms, which was confirmed by the Philippine Atmospheric, Geophysical, and Astronomical Services Administration\u0026rsquo;s regional weather forecast in Laguna for October 22, 2023 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The exact coordinates of the cacao trees in which the cacao pod isolates were collected are listed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInformation on the cacao pod collection sites. This contains the date of collection, coordinates, altitude, and weather conditions during collection.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCacao Accession Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlant ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDate of Collection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLatitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLongitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAltitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWeather\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"21\" rowspan=\"22\"\u003e \u003cp\u003e\u003cem\u003eTheobroma cacao\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003ePre-harvested\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"21\" rowspan=\"22\"\u003e \u003cp\u003eCloudy with intermittent rain showers\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.378279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e253\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.13692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.385413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e198\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.137016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.385518\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e196\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.137067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.385527\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e196\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.378233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e273\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" morerows=\"1\" nameend=\"c6\" namest=\"c4\" rowspan=\"2\"\u003e \u003cp\u003ePre-harvested\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377948\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e253\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e253\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u0026nbsp;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377791\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143756\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377799\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143734\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143725\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143725\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377677\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.143722\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.144206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.377217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.137217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.385637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e257\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.136738\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e121.385835\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCP22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/22/2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003ePre-harvested\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of\u003c/b\u003e \u003cb\u003ePhytophthora\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePathogen samples were collected from the pods of \u003cem\u003eTheobroma cacao\u003c/em\u003e L. exhibiting \u003cem\u003ePhytophthora\u003c/em\u003e blight symptoms. The collected samples were brought to the PUP Mycology Laboratory for isolation. Before isolation, the samples were surface sterilized using 70% ethanol for one minute, rinsed three times with sterile distilled water, and dried on sterile tissue paper.\u003c/p\u003e \u003cp\u003eSamples of 0.5 x 0.5 cm were excised from the cacao pericarp with partly necrotic and healthy tissues. After excision, these samples are embedded in a selective media of 20% clarified V8 agar buffered with 2 g/L calcium carbonate and amended with 100 ml/L nystatin, 200 mg/L ampicillin, and 100 mg/L rifampicin. Cultures were incubated at 25\u0026deg;C or room temperature in the dark for 2\u0026ndash;3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMorphological Characterization of the Isolates\u003c/h2\u003e \u003cp\u003eFive \u003cem\u003ePhytophthora\u003c/em\u003e isolates were used to characterize the culture and spore morphology based on their high percentage identity. Microscopic observation of cultural morphology was done using a stereomicroscope. Aseptic techniques were implemented while observing the mycelia and spores using a compound microscope. The tape method was used for observation, and the specimen was stained using lactophenol cotton blue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular Identification and PCR Amplification\u003c/h3\u003e\n\u003cp\u003eof Samples of \u003cem\u003ePhytophthora\u003c/em\u003e isolates were sentto the Department of Plant Pathology at the University of Florida for DNA extraction, amplification, and sequencing. V8 agar blocks from the colony edges of 5-day-old \u003cem\u003ePhytophthora\u003c/em\u003e isolates containing mycelia were prepared in a sterile microcentrifuge tube and then shipped to the Department of Plant Pathology at the University of Florida, following the approval of the Material Transfer Agreement and permit from the Bureau of Plant Industry in the Philippines.\u003c/p\u003e\n\u003ch3\u003eSequence and Phylogenetic Analysis\u003c/h3\u003e\n\u003cp\u003eSequence and phylogenetic analysis were performed using two bioinformatics solutions, specifically NCBI Blast and Mega X. The processed sequences sent by the Department of Plant Phytopathology, University of Florida, were converted into FASTA format. The multiple and pairwise sequence alignment of the DNA nucleotides of the different \u003cem\u003ePhytophthora\u003c/em\u003e species were rendered using MUSCLE via the MEGA program. The \u003cem\u003ePythium zingiberum\u003c/em\u003e strain (UOP389) from the NCBI GenBank is the chosen outgroup used in generating the phylogenetic tree. The best model generated using the MEGA program was chosen based on the lowest BIC scores. The phylogenetic tree was constructed using a bootstrap value of 1000 replicates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePathogenicity Test and Cross-Infection Test\u003c/h2\u003e \u003cp\u003e5 \u003cem\u003ePhytophthora\u003c/em\u003e isolates were used for the pathogenicity test on cacao and cross-infection tests on papaya and banana based on their high percentage identity. Pathogenicity and cross-infection tests were performed by artificial inoculation using a sterile 5 mm borer. Each isolate was tested in triplicate, with one sample unit per replicate. A 7-day-old \u003cem\u003ePhytophthora\u003c/em\u003e culture, grown 5 days in the dark and 2 days in light, in 2 g/L calcium carbonate-buffered 20% V8 agar was used for the inoculum blocks or agar blocks. The healthy pods and fruits were surface sterilized using 70% ethanol for one minute, rinsed with sterile distilled water twice, and blot-dried with sterile tissue paper. Using the modified protocol of Solpot \u0026amp; Cumagun (2022), a 5 mm \u003cem\u003ePhytophthora\u003c/em\u003e agar block was placed at the center of the artificially wounded cacao pods, papaya fruits, and banana fruits obtained in commercial markets [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The samples were incubated in the dark at 25\u0026deg;C. After three to seven days, successful inoculation was measured based on the extent of discoloration or lesions around the inoculation plugs by calculating the average lesion diameter in millimeters three times at different angles. Another triplicate control setup was prepared using the same 5 mm V8 agar blocks without the pathogen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe pathogenicity and cross-infection tests were conducted using a completely randomized design (CRD). The mean lesion diameter of all isolates underwent statistical analysis using the Statistical Tool for Agricultural Research (STAR), version 2.0.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bbi.irri.org/products\u003c/span\u003e\u003cspan address=\"http://bbi.irri.org/products\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and data were analyzed using Tukey's Studentized Range (HSD) test to group isolates that are not statistically different at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted with no competing interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eDr. Lourdes V. Alvarez received a Visiting Scholarship from Fulbright Philippines for the Molecular Analysis of the isolated samples at the University of Florida.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRicci Julia A. De Mesa, Myles David A. Florano, Jarvic Louis D. Suguitan, and Dr. Lourdes V. Alvarez equally contributed to this study. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge Dr. Erica Goss and the Department of Plant Pathology, University of Florida, for aiding in this research. The authors appreciate the support from Fulbright Philippines for granting Dr. Lourdes V. Alvarez a Fulbright Scholarship grant at the University of Florida.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study is available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, D., Motilal, L. \u0026amp; Origin Dispersal, and Current Global Distribution of Cacao Genetic Diversity. Cacao Diseases: A History of Old Enemies and New Encounters (eds Meinhardt, L. W. \u0026amp; Bailey, B. A.) L. W. (Springer International Publishing, (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasteel, M., Ketelaar, T. \u0026amp; Govers, F. Fatal attraction: How Phytophthora zoospores find their host. \u003cem\u003eSemin. Cell Dev. Biol.\u003c/em\u003e \u003cb\u003e148\u0026ndash;149\u003c/b\u003e, 13\u0026ndash;21 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoss, E. 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(2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubfiles.pagasa.dost.gov.ph/ncrprsd/pf.pdf\u003c/span\u003e\u003cspan address=\"https://pubfiles.pagasa.dost.gov.ph/ncrprsd/pf.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Black pod rot, Cacao, Intercropping, Pathogenicity, Phytopathology, Philippines","lastPublishedDoi":"10.21203/rs.3.rs-6390079/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6390079/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDespite its high demand, the production of cacao (\u003cem\u003eTheobroma cacao\u003c/em\u003e L.) remains low, which can be attributed to phytopathogens responsible for the destruction of cacao crops. Among these phytopathogens include \u003cem\u003ePhytophthora\u003c/em\u003e spp. - pathogenic oomycetes that target plant species through their zoospores and cause black pod rotting. This research studied the ability of \u003cem\u003ePhytophthora\u003c/em\u003e spp. to infect fruits commonly intercropped with cacao, such as papaya (\u003cem\u003eCarica papaya\u003c/em\u003e L.) and banana (\u003cem\u003eMusa\u003c/em\u003e spp.). Cacao pods exhibiting black pod rot symptoms were collected to isolate Phytophthora spp. from two cacao farms in Nagcarlan, Laguna, Philippines. Twenty-two isolates were identified as \u003cem\u003ePhytophthora palmivora\u003c/em\u003e through their culture morphology, zoospore morphology, and molecular identity through ITS gene sequencing and confirmed through phylogenetic analysis. Five selected isolates were inoculated with their original host, cacao, and cross-infected with banana and papaya, which exhibited varying degrees of black pod rot symptoms. The lesions in cacao exhibited a higher degree of disease progression compared to banana and papaya. However, statistical analysis showed that bananas were more susceptible to the infection than papayas. Quantifying the degree of infection of \u003cem\u003eP. palmivora\u003c/em\u003e from its host to commonly intercropped plants provides information on which plants are most vulnerable to infection within intercrop systems.\u003c/p\u003e","manuscriptTitle":"Cross-Infection Analysis of Phytophthora spp. Isolated from Cacao (Theobroma cacao L.) Pods at Nagcarlan, Laguna, Philippines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 17:43:48","doi":"10.21203/rs.3.rs-6390079/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"663defa1-81dc-4f58-872a-ff11c112a77b","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":48266269,"name":"Biological sciences/Plant sciences"},{"id":48266271,"name":"Biological sciences/Microbiology"},{"id":48266272,"name":"Biological sciences/Microbiology/Infectious disease diagnostics"},{"id":48266273,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2025-07-21T09:23:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-09 17:43:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6390079","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6390079","identity":"rs-6390079","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}